Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 136, Issue 4, Pages 1483-1496Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja4104217
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Funding
- NIH [GM056207, R01GM095923]
- National Science Foundation [OCI-1053575]
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The hepatitis delta virus (HDV) ribozyme catalyzes a self-cleavage reaction using a combination of nucleobase and metal ion catalysis. Both divalent and monovalent ions can catalyze this reaction, although the rate simulations to investigate the mechanism of this ribozyme is slower with monovalent ions alone. Herein, we use quantum mechanical/molecular mechanical (QM/MM) free energy and to elucidate the roles of the catalytic metal ion. With Mg2+ at the catalytic site, the self-cleavage mechanism is observed to be concerted with a phosphorane-like transition state and a free energy barrier of similar to 13 kcal/mol, consistent with free energy barrier values extrapolated from experimental studies. With Na+ at the catalytic site, the mechanism is observed to be sequential, passing through a phosphorane intermediate, with free energy barriers of 2-4 kcal/mol for both steps; moreover, proton transfer from the exocyclic amine of protonated C75 to the nonbridging oxygen of the scissile phosphate occurs to stabilize the phosphorane intermediate in the sequential mechanism. To explain the slower rate observed experimentally with monovalent ions, we hypothesize that the activation of the O2' nucleophile by deprotonation and orientation is less favorable with Na+ ions than with Mg2+ ions. To explore this hypothesis, we experimentally measure the pK(a) of O2' by kinetic and NMR methods and find it to be lower in the presence of divalent ions rather than only monovalent ions. The combined theoretical and experimental results indicate that the catalytic Mg2+ ion may play three key roles: assisting in the activation of the O2' nucleophile, acidifying the general acid C75, and stabilizing the nonbridging oxygen to prevent proton transfer to it.
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