Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 63

predicts an almost complete rescue effect. Finally, a series of single mutations
at the G5 position have been studied, including G5I, G5A, and G5D.
The general structure of the HHR active site, including identification of
indexes used to characterize key hydrogen bond networks and base stacking
interactions involving conserved residues, is shown in Fig. 2.10 . Represen-
tative hydrogen bond patterns observed in the simulations for the C3 and G8
mutants are shown in Fig. 2.11 , and for the G5 mutants are shown in
Fig. 2.12 . Averages and fluctuations for key indexes used to characterize
the active site are listed in Tables 2.8 and 2.10 for the reactant state and
Tables 2.9 and 2.11 for the activated precursor state. Table 2.12 lists indexes
used to characterize the base stacking interactions between G8 and C1.1 in
the wild type, benign U7C and G8I single mutants, and double mutant sim-
ulations. A summary of the overall mutational effects inferred from the MD
simulations are provided in Table 2.13 and compared to experimental values
for the relative catalytic rates.
In the following discussion, we will apply certain mechanistic assump-
tions in our analysis, in particular regarding the role of G12 and G8
2 0 OH as the general base and acid, respectively. These roles are supported
by structural data, 73,74 mutagenesis, 61,68 and biochemical 59,60 studies, but
have not been definitively proven. Assuming this plausible mechanistic
hypothesis, we then ask whether our simulation results can explain the ori-
gin of the mutational effects. It should be emphasized that the mechanistic
assumptions regarding G12 and G8 be incorrect, so must be the interpreta-
tions of the simulation data that invoke this model. Further study is needed
to resolve these details, for example, using molecular simulation of the cat-
alytic chemical steps of the reaction with combined QM/MM methods.

5.1. Results
5.1.1 Control simulations: wild type and U7C
As a precursor to the discussion of the origin of mutation effects on reaction
rate, a characterization of the key elements of the WT simulation that affect
catalysis is needed. Moreover, to lend credence to our simulation method-
ology and our mechanistic interpretation of simulation results, we perform
a control simulation of a U7C mutation that has been observed experi-
mentally to have no adverse effect on the relative rate of reaction as
determined by the ratio of rate constants for WT and mutant reactions,
k rel k mut / k wt . 151 The WT and U7C control simulation results are included

in all of the tables for reference and comparison.

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