Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 77

of any of the key hydrogen bonds or in-line fitness indexes. However, a
closer examination reveals some differences in the base stacking of C3U/
G8D relative to theWT and control simulations ( Table 2.12 ). The alteration
of the relative position between C1.1 and G8, in particular the increase in
the r 7 stacking distance, shifts the position of the A9 phosphate leading to the
modest increase in d 0 . Consequently, the simulation results predict this dou-
ble mutation to have a significant rescue effect, but not necessarily fully res-
cue to the WT activity.

5.1.4 Single mutations at the G5 position
The G5 position forms an important element in the structural scaffold of the
active site that helps hold the scissile phosphate in position via hydrogen
bond interactions with the sugar and base functional groups of C17, and
with the nucleobase of A14 ( Fig. 2.10 ). These include hydrogen bonds
between the G5:N1 endocyclic amine and C17:O2 ( r 4 ), and the G5:N2
exocyclic amine and C17:O4 0 ( r 5 ) and A14:N1 ( r 6 ). Here, we consider a
series of single mutations (G5I, G5A, and G5D), representative hydrogen
bond patterns for which are shown in Fig. 2.12 .

5.1.4.1 G5I mutation retards the general acid step
Experimental rate measurements for the G5I mutation suggest a reduction of
the catalytic rate by 0.006 94 to < 10 3 , 96 respectively. This mutation elim-
inates the exocyclic amine of guanine, and hence the hydrogen bond with
C17:O6 and A14:N1. As a result, in the reactant state ( Table 2.10 ), the
integrity of the active site is somewhat disrupted ( d 0 increases by 0.67 ˚ ).
In the activated precursor state ( Table 2.11 ), the hydrogen bond between
G8:H2 0 and C1.1:O5 0 is lost ( r HA 3.6 ˚ ), which would severely impair

¼

the general acid step of catalysis.

5.1.4.2 G5A mutation severely disrupts the active site integrity and in-line
fitness
The G5Amutation leads to no detectable activity in the HHR. 83 This muta-
tion eliminates both the endocyclic and exocyclic amines at the 1 and 2 posi-
tions of guanine that hydrogen bond with C17 and A14, and replaces the O6
carbonyl group with a new exocyclic amine at the 6 position. The result
( Table 2.10 ) is that the nucleobase shifts such that the exocyclic amine at
the 6 position of inosine hydrogen bonds with C17:O4 0 , previously hydro-
gen bonded to the endocyclic amine at the 1 position. The other hydrogen