Evidence for the Role of Active Site Residues in the Hairpin Ribozyme from Molecular Simulations along the Reaction Path

The hairpin ribozyme accelerates
a phosphoryl transfer reaction
without catalytic participation of divalent metal ions. Residues A38
and G8 have been implicated as playing roles in general acid and base
catalysis, respectively. Here we explore the structure and dynamics
of key active site residues using more than 1 μs of molecular
dynamics simulations of the hairpin ribozyme at different stages along
the catalytic pathway. Analysis of results indicates hydrogen bond
interactions between the nucleophile and proR nonbridging oxygen are
correlated with active inline attack conformations. Further, the simulation
results suggest a possible alternative role for G8 to promote inline
fitness and facilitate activation of the nucleophile by hydrogen bonding,
although this does not necessarily exclude an additional role as a
general base. Finally, we suggest that substitution of G8 with N7-
or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5′
leaving group position, would provide valuable insight into the specific
role of G8 in catalysis.