Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 46

simulated free-energy value for the transition state of the general acid step in
Fig. 2.5 C is 13.7 kcal/mol with respect to the activated precursor state. The
intermediate is only 6.7 kcal/mol lower in free energy than the activated pre-
cursor, and has a 20.4 kcal/mol barrier to break down into the product state
with a proton transferred to the C1.1:O5 0 leaving group. We note that this
barrier is effectively reduced if proton-tunneling effects are included.
3.2. Summary
The QM/MM free-energy profile work presented here explores a specific
mechanistic scenario, departing from the activated precursor state, which
assumes the catalytic metal ion is in a position bridging the A9 and scissile
phosphates, and the 2 0 OH of G8 acts as a general acid catalyst. The former
metal ion-binding mode has not yet been observed experimentally, but has
been inferred from biochemical experiments, and predicted by molecular
simulations summarized in Section 2 . The QM/MM results provide further
computational support for the plausibility of a cleavage mechanism where
phosphoryl transfer and general acid catalysis are stepwise, and that the cat-
alytic divalent metal ion plays an active role in the chemical steps of catalysis.
The results of these QM/MM simulations are of interest to compare with
very recent reports that use thio/Cd 2 þ -rescue experiments to probe the gro-
und state coordination of a catalytic metal ion to the scissile phosphate of an
eHHRmotif 122 in a bridging mode identical to that proposed in the current
theoretical investigation. It is thus of great interest to extend our future the-
oretical studies to be more directly comparable to these experiments.
3.3. Simulation setup and protocol
The free-energy simulations performed here use the same settings and pro-
tocols as the QM/MM simulations in Section 2.3.2 , with the following
additions and exceptions: The QM region includes G8, A9, C1.1, C17,
and an Mg 2 þ ion with coordinated water molecules (81 atoms).
For Mg 2 þ , the AM1/d model parametrized by Imhof was utilized. 123 For
the MM region, the all-atom AMBER parmbsc0 force field was
employed, 124 along with the TIP4P-Ewald water model 125 and the consis-
tent set of monovalent ion parameters. 126 Multidimensional PMF profiles
were generated along reaction coordinates corresponding to phosphoryl
transfer, proton transfer from the general acid to the leaving group, and diva-
lent metal ion-binding mode.