Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme

Abstract

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 pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme 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.

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

“…Assuming that the substitution is non-perturbing and that the difference in activity is solely due to the different extent of base deprotonation, a pK a of 10.6 can be calculated for G8, in agreement with our assumption that G8 has a pK a > 10. Hence, the data of Liu et al (2009) are entirely consistent with the hypothesis that G8 participates in general acid-base catalysis.…”

“…It is interesting to note that the pK a of 8-azaguanine is z1 pH unit lower than that of guanine (Liu et al 2009). Substitution of 8-azaguanine for guanine would therefore be expected to increase f B at pH 8 since a greater proportion of molecules would have a deprotonated base.…”

“…Recently Liu et al (2009) directly measured the ionization state of 8-azaguanine substituted at position 8 by means of the increased fluorescent emission when the base becomes deprotonated. Under standard conditions, the intrinsic pK a of 8-azaguanine at position 8 of an active hairpin ribozyme was 9.5.…”

The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine

“…8azaAHp Rz and 8azaAHpm Rz RNAs exhibited a 40-fold lower quantum yield than 8azaAMP. RNAs with 8azaG substitutions exhibited a similar level of fluorescence quenching relative to 8azaGTP (19,33). Quenching of 8-azapurine fluorescence in RNAs is likely attributable to orbital overlap with flanking nucleobases as characterized previously for 2-aminopurine in B-form helices (34 -36).…”

“…In contrast, Hp Rz with an abasic linkage in place of G8 exhibit an 850-fold loss of self-cleavage activity, but the apparent pK a for the reduced activity remains near 6 (18). Our recent evidence that the microscopic pK a values are above 9 for 8-azaguanosine (8azaG) ionization in 8azaGHp Rz and do not correlate with the pK a(app) values below 7 obtained from 8azaGHp Rz pH-rate profiles also argues that the pH-dependent step in the Hp Rz reaction reflects the protonation state of A38 rather than G8 (19).…”