Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 44

Table 2.4 Relative free energies and internuclear distances at various states of RNA
self-cleavage in HHR

Reactant TS1 Intermediate TS2 Product

C17:O2 0 d P 3.50 (04) 1.76 (05) 1.66 (03) 1.67 (03) 1.68 (03)

P d C1.1:O5 0 1.65 (03) 2.11 (05) 4.51 (04) 4.24 (48) 3.63 (23)

G8:O2 0 d H

0.96 (00) 0.96 (00) 0.96 (00) 1.78 (04) 3.75 (04)

H d C1.1:O5 0

2.57 (51) 4.07 (47) 4.13 (73) 1.03 (03) 1.00 (03)

Mg 2 þ d C1.1:O5 0 3.99 (18) 3.61 (17) 2.02 (05) 2.83 (86) 4.48 (05)

Mg 2 þ d G8:O2 0

4.56 (18) 4.03 (18) 4.33 (06) 3.38 (86) 2.03 (05)

G 0.0 (4) 24.4 (6) 6.7 (3) 13.7 (7) 13.6 (9)

△

Free energies ( △ G ) are in kcal/mol, which were extracted from 1D PMF profiles along the minimum
free-energy path in the 2D profiles. Average distances ( X Y ) are in ˚ . Standard deviations are listed in
parentheses divided by the decimal precision of the average values.

Mg 2 þ -binding mode as a second dimension. Table 2.4 summarizes key
average geometrical parameters and free-energy values for stationary points
along the minimum free-energy reaction paths derived from the two
separate 2D PMF profiles, which are the dark arrow in Fig. 2.5 B and the
white arrow in Fig. 2.5 C, respectively.

3.1.2 Phosphoryl transfer is rate limiting and facilitated by electrostatic
stabilization by Mg 2þ
Figure 2.5 B depicts the 2D free-energy profile for the Mg 2 þ -binding mode
during the phosphoryl transfer step. Comparing to the free-energy barrier in
another 2D PMF profile for the general acid catalysis ( Fig. 2.5 C), we con-
clude that this phosphoryl step is rate controlling. To further refine the sam-
plings in MD simulations, we generated a 1D PMF profile following the
minimum free-energy reaction path ( Fig. 2.5 B). The computed free-energy
barrier for this rate-controlling step is approximately 24.4 kcal/mol.
The minimum free-energy reaction path indicates that the position of
the Mg 2 þ ion follows the negative charge along the phosphoryl transfer
reaction coordinate, in order to provide electrostatic stabilization. The
change in the Mg 2 þ position is continuous and monotonic throughout
the phosphoryl transfer step ( Fig. 2.5 B), although it is most pronounced
in the initial and final stages when the nucleophile and leaving group have
the greatest negative charge. The transition state is late ( Table 2.5 and
Fig. 2.5 ), having a P d C1.1:O5 0 distance of 2.11 ˚ . As the P d C1.1:O5 0