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Probing general acid catalysis in the hammerhead ribozyme.
Jason M Thomas1, David M Perrin
1Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada, V6T 1Z1.
Journal of the American Chemical Society
|January 22, 2009
Summary
Divalent metal cations (M(2+)) and a specific ribozyme group are crucial for hammerhead ribozyme general acid catalysis. This study clarifies their roles, showing M(2+)-cofactor coordination lowers the G8 2'-OH pK(a).
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Hammerhead ribozymes are RNA enzymes with a catalytic core.
- Recent studies suggest specific ribozyme groups and metal ions are involved in catalysis.
- The precise catalytic role of divalent metal cations (M(2+)) in hammerhead ribozymes remains unclear.
Purpose of the Study:
- To functionally characterize the general acid catalysis mechanism in the S. mansoni hammerhead ribozyme.
- To elucidate the role of divalent metal cations (M(2+)) as cofactors in this catalytic process.
- To investigate the contribution of the G8 2 extquotesingle-OH ribozyme residue to general acid catalysis.
Main Methods:
- Comparative analysis of hammerhead ribozyme cleavage on natural ribo-phosphodiester and bridging-5 extquotesingle-phosphorothioate substrates.
- Site-directed mutagenesis of the G8 2 extquotesingle-OH ribozyme residue.
- Substitution of divalent metal cations (M(2+)) cofactors.
- Synthesis of radiolabeled bridging-5 extquotesingle-phosphorothioate substrates.
Main Results:
- Cleavage of natural substrates was inhibited by G8 2 extquotesingle-OH modification and M(2+) cofactor presence/identity.
- Cleavage of phosphorothioate substrates was insensitive to these modifications.
- General acid pK(a) perturbation was observed upon M(2+) substitution and G8 2 extquotesingle-OH modification.
Conclusions:
- Both M(2+) cofactors and the G8 2 extquotesingle-OH are essential for hammerhead ribozyme general acid catalysis.
- M(2+) cofactors do not stabilize the leaving group via Lewis acid interactions.
- Transition state M(2+) coordination of G8 2 extquotesingle-OH likely lowers its pK(a), enhancing proton transfer to the leaving group.
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