Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 45

Table 2.5 Index of simulations performed in Section 4

Mg 2þ
position

Simulation
(ns)

Analysis
(ns) State of C17:2 0 OH

Abbr.

RT-C-Mg Neutral reactant C-site 300 250

RT-B-Mg Neutral reactant B-site 300 250

dRT-
C-Mg

Deprotonated
precursor

C-site 1 -

<

dRT-
B-Mg

Deprotonated
precursor

B-site 300 250

RT-Na Neutral reactant Absent 300 250

dRT-Na Deprotonated

precursor

Absent 300 250

Each simulation is given an abbreviation (Abbr.) that is referenced in the text. The simulations differ in
the protonation state of the 2 0 OH nucleophile (State of C17:2 0 OH) being either in the “neutral reactant”
or “deprotonated precursor” state. The simulations also differed from the presence/absence and initial
placement of the Mg 2 þ ions in the active site. In the presence of a Mg 2 þ ion in the active site, initial
placement could be in the “C-site” coordinated to O2P of the A9 phosphate and N7 of G10.1, or in
the “B-site” position bridging between the O2P atoms of the A9 and scissile phosphates. In the
“dRT-C-Mg” simulation of the deprotonated precursor with Mg 2 þ initially placed in the C-site, the
Mg 2 þ ion quickly migrated to the B-site position, and exhibited dynamics essentially equivalent to
the dRT-B-Mg simulation, and thus was not carried out further or analyzed. All simulations were carried
out in 0.14 MNaCl at 298 K and 1 atm pressure, and with the exception of the “dRT-C-Mg” simulation
mentioned above, were carried out to 300 ns, the last 250 ns of which was used for analysis.

bond breaks, the Mg 2 þ ion forms an inner-sphere coordination, leading to a
Mg 2 þ -bound O5 0 alkoxide intermediate.

3.1.3 General acid catalysis is concerted with changes in
Mg 2þ -binding mode
The 2D free-energy profile for the Mg 2 þ -binding mode during the general
acid catalysis is shown in Fig. 2.5 C. The general acid step considered here
assumes that the 2 0 OH of G8 acts as a general acid catalyst to transfer a proton
to the C1.1:O5 0 leaving group. Table 2.5 and Fig. 2.5 C indicate that proton
transfer occurs after the formation of Mg 2 þ -coordinated cleaved intermedi-
ate, but that proton transfer is concerted with changes in the Mg 2 þ -binding
mode. Unlike the phosphoryl transfer step, participation of the Mg 2 þ along
the reaction coordinate is most pronounced not at the endpoints of the step
but near the midpoint where the proton transfer occurs.
To further enhance the samplings in MD simulations, we generated a
1D-PMF profile following the minimum free-energy reaction path. The