Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 27

mammalian messenger RNA, suggesting HHR's possible common role in
posttranscriptional gene regulation. 56
However, the detailed reaction mechanism of HHR is still elusive
despite significant experimental and theoretical work. 38,39,41,44,57-61 One
aspect of the catalytic mechanism that has perplexed the community
involves the specific role of divalent metal ions in catalysis. Specifically,
one of the main puzzles involves the apparent inconsistency between the
interpretation of thio and metal ion substitution 62-66 and mutational 61,67-69
experiments with available crystallographic structural information of the
minimal HHR (mHHR) sequence. 70-72 Biochemical experiments have
been interpreted to suggest that a pH-dependent conformational change
must precede or be concomitant with the catalytic chemical step, including
a possible metal ion bridge between the A9 and scissile phosphates. This is
inconsistent with the interpretations of crystallographic data for the mHHR
motif, where A9 and scissile phosphates are found to be about 20 ˚

apart. Moreover, the function of the 2 0 -OH group of G8 remains
unclear from the data. 38,41 Recent crystallographic studies of the extended
HHR (eHHR) have characterized the ground state active site architecture 73
and its solvent structure, 74 including the binding mode of a presumed
catalytically active divalent metal ion in the active site. These findings,
together with molecular simulation studies, 75-79 have reconciled a long-
standing controversy between structural and biochemical studies for this
system. 80
In this chapter, we summarize our recent efforts to unveil the detailed
mechanisms of HHR catalysis, with emphasis on the characterization of
metal ion-binding modes and their relationship with structure and catalysis.
Through molecular dynamics (MD) simulations, we first examined metal
ion-binding modes in the HHR at various stages of progression along
the reaction coordinate, and the characterization of the electrostatic envi-
ronment of the HHR and its ability to recruit cationic charge, as well as
the relationship between the threshold occupancy of metal ions and the
formation of catalytically active conformations. Using QM/MM tech-
niques, we identified the most plausible reaction path of the HHR enzy-
matic reaction, and the correlation between the metal ion coordination
and the reaction path. To further clarify and verify the proposed mecha-
nisms, a systematic computational mutagenesis study of HHR on three
key residues was performed to provide atomic-level explanation of
experimentally observed mutational effects, as well as prediction of possible
rescuing mutations.

70-72