Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 75

5.1.2.3 G8A mutation blocks the general acid step in the activated
precursor
The catalytic rate of the G8A mutant is reduced to less than 0.004. 83 Sim-
ulation results indicate that G8A mutation considerably weakens the base
pair with C3 such that only one weak hydrogen bond remains intact
( Fig. 2.11 and Table 2.8 ). In the reactant state simulation, G8A does not
appear to dramatically alter the active site contacts relative to the WT
simulation, with the exception that A8:N1 d C3:N3 distance increases
due to a shift in the hydrogen bond pattern.
In the activated precursor state ( Table 2.9 ), however, the hydrogen bond
between G8:H2 0 and C1.1:O5 0 is significantly disrupted relative to the WT
simulation. The breaking of this key hydrogen bond ( r HA ¼ 4.13 ˚ ) in the

G8A mutation and movement of the general acid away from the leaving
group effectively block the general acid step of the reaction. The G8Amuta-
tion has the largest effect on catalysis of all the single mutations at the G8
position considered here.

5.1.2.4 G8D mutation alters the active site structure and weakens the
interaction of the nucleophile and general base
Mutation of G8 to 2-aminopurine 61,96 or to 2,6-diaminopurine (G8D) 96
reduces the reaction rate by approximately three orders of magnitude.
The effect of this mutation in the reactant state is to weaken the hydrogen
bonding between G8D and C3 relative to theWT simulation. This results in
corruption of the active site structure ( d 0 increases by 2.89 ˚ relative to the
WT simulation) and impairs hydrogen bonding of the 2 0 OH nucleophile
with the general base.

5.1.3 Double mutations at the C3/G8 positions
In order to better understand the role played by individual residues on ribo-
zyme structure and function from mutagenesis data, it is useful to consider
the effects of double mutations that are able to restore, at least in part, activity
lost by deleterious single mutations. Here, we consider a series of double
mutations (C3G/G8C, C3U/G8A, and C3U/G8D), for which representa-
tive hydrogen bond patterns are shown in Fig. 2.11 .

5.1.3.1 C3G/G8C double mutation has a partial rescue effect
The C3G/G8C double mutation preserves strong hydrogen bond interac-
tions between the 3 and 8 positions and has a partial rescue effect relative to