Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 31

for catalysis. In the first eHHR structure, no divalent metal ions were
resolved, 73 and this left the burning question as to what was the specific role
of divalent metal ions in catalysis. Moreover, the roles of G8 and G12 as
plausible candidates as the general acid and the general base, respectively,
as supported by their positions in the active site, still lacked compelling evi-
dence as to how the ribozyme environment might induce the required
shifts in p K a values to be catalytically active. For example, in order for
G12 to act as a general base, it must be deprotonated at the N1 position
(solution p K a of 9.2). Further, the crystal structure revealed that it was
the 2 0 OH of G8 that was positioned to act as a general acid, which has
been estimated to have considerably elevated p K a values ( > 14). Conse-
quently, the p K a values of the implicated functional groups would require
considerable shifts toward neutrality to be consistent with the apparent p K a
values derived from pH-rate profiles. Therefore, a detailed investigation of
the roles of metal ions and the microenvironment around the active site is
desperately needed.
An initial theoretical study was communicated 75 that offered a prediction
that a divalent metal ion could stably occupy a position bridging the A9 and
scissile phosphates in the transition state of the eHHR. A subsequent joint
crystallographic and molecular simulation study of the ground state eHHR 74
revealed a single Mn 2 þ bound directly to the A9 phosphate in the active site,
accompanying a hydrogen bond network involving a well-ordered water
molecule spanning N1 of G12 (the general base) and 2 0 OH of G8 (previ-
ously implicated in general acid catalysis). The crystallographic data for
the ground state solvent structure, however, did not show a divalent ion
in the bridging position in the transition state suggested by simulation.
2.3. Probing metal ion-binding modes in HHR with
molecular simulation
In the first eHHR crystal structure, the two presumably negatively charged
oxygen atoms, A9:O2P and C1.1:O2P, are around 4 ˚ away, an ideal dis-
tance for direct coordination of a Mg 2 þ ion in a bridging “B-site” position.
Hence, placing a divalent metal ion between them would be a reasonable
first attempt to explore the possible roles of the required metal ion,
although there is no metal ion resolved in the first eHHR crystal structure. 75
Nevertheless, the solvent structure of eHHR 74 suggests a different site for
Mn 2 þ , which is defined as the “C-site” ( Fig. 2.2 ). Hence, the first set of
simulations 75,76 was performed to explore the possible positions and roles
of the Mg 2 þ .