Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 68

Table 2.8 Characterization of the active site structure and fluctuations for the C3 and G8
mutants in reactant states—cont'd

C3U/
G8A

C3U/
G8D

C3G/
G8C WT U7C C3U G8A G8I G8D

y 3 163.5

(8.8)

164.4
(8.3)

162.1
(9.5)

149.3
(14.8)

162.7
(9.6)

163.8
(8.9)

- 155.9

(10.4)

161.3
(10.2)

This table lists key structural indexes fluctuations for the C3 and G8 mutants, along with the control
mutant U7C in reactant states. Data analysis was performed over the last 65 ns of each simulation
with a 10 ps sampling frequency. Distance and angles ( Fig. 2.10 ) are in ˚ and degrees, respectively.
SDs are listed in parentheses. Boldface font is used to highlight key quantities that are significantly al-
tered with respect to the wild-type (WT) simulation upon mutation and that are discussed in the text.
F is the in-line fitness index. 150 The r NN distance is between nucleobases in the 3 and 8 positions. r HB
and y HB are the hydrogen bond length and angle for the general base step; defined by G12:N1 d C17:
HO2 0 d C17:O2 0 . r HA and y HA are the hydrogen bond length and angle for the general acid step;
defined by C1.1:O5 0 d G8:HO2 0 d G8:O2 0 . The hydrogen bond contact percentage for the above
entries, defined as the percentage of the snapshots in which r 3.0 ˚ and y 120 . The hydrogen bond
contact percentage for the above entries, defined as the percentage of the snapshots in which r 2.5 ˚ and
y 150 .

From this mechanistic picture, several conditions for catalytic compe-
tency of the HHR can be inferred. First, the general base must be correctly
positioned to abstract a proton from the nucleophile to form the activated
precursor. Second, the structure of the active site must allow the
activated nucleophile to be in-line with the scissile phosphate, and fluctua-
tions must sample conformations that have a high degree of in-line fitness.
Third, the integrity of the active site, and in particular, the proximity of the
A9 and scissile phosphates must be conducive to binding a bridging
divalent metal ion. Fourth, the general acid must be poised to donate a
proton to the leaving group to facilitate cleavage. In order to satisfy these
conditions, a specific network of hydrogen bonds and base stacking interac-
tions must be in place. Indexes correlated with each of these conditions are
depicted in Fig. 2.10 . The average values and fluctuations of these
indexes for the WT simulation are listed in the tables for reference, and
representative hydrogen bond networks involving conserved residues are
shown in the figures.

5.1.1.2 U7C control simulation satisfies all of the conditions
for WT catalysis
The U7C mutant has a catalytic rate virtually identical to that of the WT
( k rel 1.1). 147 Comparison of the WT and U7C control simulations shows

no major differences in the indexes likely to be key for catalysis
( Tables 2.8-2.13 ). The general base forms a stable hydrogen bond with

¼