Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 79

Thus, the mutants that exhibit large shifts in the relative positions of key
residues in the active site scaffold and that are locked by hydrogen bonding
networks (i.e., C3U, G8D, and G5D) generally display the largest disrup-
tions of the active site architecture. Alternately, mutants that do not exhibit
shifts of the relative positions of residues (i.e., G8I, C3U/G8A, and C3G/
G8C) or that have weakened hydrogen bond networks (G8A, G5A, and
G5I) have d 0 values more similar to the WT and control simulations.

5.2.2 In the activated precursor state, strong hydrogen bond
networks stabilize the active site architecture
In all of the activated precursor simulations, the relative positions of the A9
and scissile phosphates are very similar ( d 0 values of around 3.0 ˚ ). This is a
result of the bridging Mg 2 þ ion that locks the A9:O2P and C1.1 O2P posi-
tions. The general base indexes are also similar between the different simu-
lations due to the strong hydrogen bond that forms between G12:N1 and
the deprotonated C17:O2 0 nucleophile. The one exception is for d-G5D,
where the loss of a hydrogen bond between D5 and the C17:O4 0 causes
the position of the sugar to shift and the hydrogen bond between the acti-
vated nucleophile and G12 to be less populated.
The orientation of the implicated general acid, G8:O2 0 , which is poised
to donate a proton to the C1.1:O5 0 leaving group, is important for catalytic
competency. Alterations in the hydrogen bond network that disrupt the
position of G8:O2 0 , particularly the base pair hydrogen bonds between
C3 and G8, will have an adverse effect on the general acid step. These alter-
ations can be tracked by monitoring the hydrogen bond distance r HA
( Tables 2.9 and 2.11 ) and indicate that activated precursor simulations gen-
erally have similar general acid indexes ( r HA and HA ) with a few notable

exceptions. In the d-G8A simulation, the C3-G8 hydrogen bond network
is lost. In the d-C3U/G8D simulation, the C1.1-G8 base stacking is signif-
icantly different from other mutants. In the d-G5I and d-G5A simulations,
the hydrogen bond network between G5 and C17 is disrupted.
Hence, in the activated precursor state, the two strands are brought
together by the bridging divalent metal ion and a strong C3-G8 hydrogen
bond network is necessary to keep G8 in position so that the general acid
(G8:O2 0 ) is poised for catalysis. The interaction between G5 and C17 is
important since G5 stabilizes the orientation of both the nucleobase and
ribose of C17, and it helps position the nucleophile in-line with the scissile
phosphate. Disruption of G5-C17 hydrogen bonds will either prevent
in-line attack (d-G5A and d-G5D) or alter the position of the general acid

y