We report results from combined quantum mechanical/molecular mechanical (QM/MM) free energy simulations to explore metal-assisted phosphoryl transfer and general acid catalysis in the extended hammerhead ribozyme. The mechanisms considered here assume that the 2'OH group of C17 has already been activated (i.e., is deprotonated) and acts as a nucleophile to go on an in-line attack to the adjacent scissile phosphate, passing through a pentavalent phosphorane intermediate/transition state, followed by acid-catalyzed departure of the O5' leaving group of C1.1. A series of six two-dimensional potential of mean force profiles are reported in this study, requiring an aggregate of over 100 ns of QM/MM simulation. The simulations employ the AM1/d-PhoT semiempirical quantum model and linear-scaling QM/MM-Ewald method and explore mechanistic pathways for the self-cleavage. Results support the plausibility of a cleavage mechanism where phosphoryl transfer and general acid catalysis are stepwise, and where the catalytic divalent metal ion plays an active role in the chemical steps of catalysis.
