Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 78

bonds to C17 and A14 are eliminated. This results in considerable degrada-
tion of the active site ( d 0 increases by 0.98 ˚ in the reactant state), poor
in-line fitness in the activated precursor state ( F decreases by 50%), and
greatly reduces hydrogen bonding interaction between G8:H2 0 and C1.1:
O5 0 ( r HA increases by 1.1 ˚ ). Overall, the active site structure and position-
ing of the general acid in the activated precursor are not conducive for
catalysis.

5.1.4.3 G5D mutation severely disrupts the active site integrity and in-line
fitness
The G5Dmutation leads to reduction of catalytic rate by four orders of mag-
nitude. 96 The hydrogen bond pattern for this mutation is not as severely
perturbed as that for the G5A mutation. Nonetheless, in the reactant state
( Table 2.10 ), the average distance between the A9 and the scissile phosphates
( d 0 ) increases to 5.2 ˚ , and the hydrogen bond between the 2 0 OH nucleo-
phile and G12:N1 (the implicated general base) is less pronounced. In the
activated precursor state ( Table 2.11 ), the general base hydrogen bond is dra-
matically weakened ( r HB 2.62 ˚ ), and the average in-line attack angle, ¼

y incl , is reduced to 139.7 degrees. These structural deviations are expected

to hinder the attack of the nucleophile on the scissile phosphate.

5.2. Discussion
5.2.1 In the reactant state, shifted residue positions stabilized by
hydrogen bond networks disrupt the active site integrity
Simulation results ( Table 2.8 ) show that among the C3 and G8 mutants, d 0
exhibits the greatest deviation for C3U ( d 0 6.64 ˚ ) and G8D ( d 0 5.86 ˚ )

¼ ¼

3.97 ˚ )

compared to the WT ( d 0 ¼ and U7C control simulation

4.27 ˚ ). In these two mutants, the C3/G8 base pairs are held together
by two hydrogen bonds and the bases are shearer relative to the WT
( Fig. 2.11 ). In other mutants, including C3U/G8A, G8I, and C3G/G8C,
all d 0 values are similar to WT and U7C. In the case of the C3U/G8D dou-
ble mutation, subtle changes in base stacking produce a slight shift in the rel-
ative positions of C1.1 and G8 ( Table 2.12 ), and this deviation is stabilized
by a strong hydrogen bond network leading to a modest increase in d 0
( d 0 ¼

( d 0 ¼

4.68 ˚ ). In the case of mutations at G5, the G5Dmutant has the largest
value for d 0 ( d 0 5.2 ˚ ). The G5D mutant also displays a significant shift in

the orientation of D5 with respect to C17 ( Fig. 2.12 ) that is stabilized by a
fairly strong hydrogen bond network.

¼