Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 76

the C3G and G8C single mutations. It, nonetheless, still reduces the activity
150-200-fold relative to theWT. 70,149 The purine/pyrimidine substitutions
alter the local stacking environment and lead to subtle structural changes in
the active site. As a consequence, the average distance between the A9 and
scissile phosphates increases by 0.29 ˚ relative to the WT simulation, and
the hydrogen bond between the 2 0 OH nucleophile and general base is
weakened ( r HB decreases from 2.07 ˚ in the WT to 2.50 ˚ in the C3G/

G8C mutant).

5.1.3.2 C3U/G8A double mutation is relatively benign
The C3U/G8A double mutation is an isosteric substitution that slightly
weakens the base pair hydrogen bonding, and it has a relatively benign
effect on activity ( k rel around 0.5-0.012). 67,149 The simulation results
for C3U/G8A are similar in hydrogen bond pattern to the relatively
benignG8IsinglemutationandtheWTbasepair.TheC3U/G8Amuta-
tion is a slightly larger perturbation than G8I, and the C3U/G8A simula-
tion results indicate correspondingly more significant deviations from the
WT. The C3U/G8A double mutation is overall similar to the C3G/G8C
double mutation. Although the pyrimidine/purine pattern is reversed in
C3G/G8C, it preserves the same number of base pair hydrogen bonds
as the WT. The average distance between the A9 and scissile phosphates
increases by 0.15 ˚ in the C3U/G8A mutant relative to the WT simula-
tion, and the average hydrogen bond distance between the 2 0 OH nucle-
ophile and general base is increased by 0.37 ˚ .Thisisexpectedtoonly
moderately retard the efficiency of the general base step of the reaction.
No other notable effects on the structure or dynamics were observed as
a result of this mutation.

5.1.3.3 C3U/G8D double mutation is predicted to have a rescue effect
The results of the hydrogen bond-preserving C3G/G8C double mutation
and isosteric C3U/G8A double mutation both show partial to strong rescue
effects on HHR activity. Computationally, we performed simulations of an
isosteric C3U/G8D double mutation that also preserved the number of
native base pair hydrogen bonds. Despite the corresponding single muta-
tions having significant deleterious effects on activity, the double mutation
is predicted by our simulations to have a rescue effect.
The only significant deviation from theWTwas in d 0 in the reactant state
simulation, which increased by around 0.7 ˚ , but did not lead to disruption