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Active Site Purines and Catalysis of RNA Self‐cleavage
Author(s) -
Fedor Martha J.,
Liu Lu,
Cottrell Joseph W.,
Scott Lincoln G.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.412.1
Subject(s) - deprotonation , chemistry , ribozyme , protonation , hairpin ribozyme , active site , guanosine , catalysis , stereochemistry , deoxyribozyme , vs ribozyme , nucleotide , photochemistry , rna , biochemistry , organic chemistry , dna , ion , gene
The hairpin ribozyme mediates catalysis through nucleotide functional groups, without metal cation cofactors. The positions of G8 and A38 in the ribozyme active site resemble the orientation of two histidines in the active site of ribonuclease A, leading to the model that G8 and A38 mediate catalysis through a similar general acid base mechanism. However, adenosine and guanosine undergo ionization only at pH extremes, at least in solution, which seems to make them poor acid base catalysts. We used 8‐azapurine fluorescence to learn whether purine ionization equilibria change in the active site relative to pK a values in solution. 8‐azapurines display high fluorescence emission intensity when N1 is deprotonated and low intensity when N1 is protonated. A ribozyme with 8azaG8 exhibits full catalytic activity and cleaves with an apparent pK a value in the neutral range, similar to an unmodified ribozyme. Microscopic pK a values for deprotonation of 8azaG 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 self‐cleavage activity with increasing pH does not reflect G8 deprotonation and G8 is mostly protonated at neutral pH. A simple interpretation of these results is that G8 functions in the protonated form, perhaps by donating hydrogen bonds that stabilize the transition state.