288 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
hammerhead RNAs at the same pH 6. The reference 55 researchers also found
that substitutions at the 2 ′ - OH position at the cleavage site with either 2 ′ -
OCH 3 or 2 ′ - F resulted in crystal structures in which the conformational change
leading to the cleavage reaction would not take place. Based on their observa-
tions, the Scott group concluded that a pH - dependent conformational change
is the rate - determining step for hammerhead self - cleavage reactions and that
an ionizable proton at the 2 ′ - position is required to achieve the necessary
conformation. They hypothesized then that deprotonation at the cleavage - site
ribose 2 ′ - OH drives the needed conformational change.
In 2003, Victoria DeRose published a Current Opinion in Structural Biology
paper that discussed the known information on metal ion binding to catalytic
RNA.^16 Much of the foregoing discussion of the hammerhead ribozyme and
the cleavage reaction it catalyzes has been summarized in this paper. DeRose
noted that cations provide chemical functionalities not available in the RNA
itself and may be necessary for ribozyme catalysis. However, if cations are
indeed required, the challenge is to assign their role as necessary for structure
(RNA folding) or for catalytic enhancement of the chemical reaction carried
out by the ribozyme. DeRose would like to defi ne the metal ion involved in
catalysis as one that (1) stabilizes the reaction ’ s transition state and (2) is
associated with a group that changes bond order during the catalytic reaction.
Spectroscopic analyses —^31 P NMR or EPR studies using paramagnetic Mn 2+ ,
for instance — have, in rare instances, pinpointed the metal ’ s location but more
often have shown the overall infl uence of metal ions on folding. However,
analytic methods have been less successful in identifying metal - dependent
changes in ribozyme catalytic activitynot dependent on folding (conforma-
tional change). One biochemical method that comes close to accomplishing
this has been the phosphorothioate metal - rescue (PS - rescue) experiment dis-
cussed previously. Applying DeRose ’ s criteria, one would say that a metal site
identifi ed by PS - rescue to be far away (and stay far away) from the active site
is defi ned as structural. Conversely, if a PS - rescued metal site involves a bond
that changes order during the catalyzed chemical reaction then that metal ion
may be directly involved in catalysis.
One would like to identify such a site in the hammerhead ribozyme –
substrate complex. The A 9 /G 10.1 metal binding site shows possibilities. X - ray
crystallography (PDB: 300D) shows an Mn 2+ – A 9 nonbridging phosphate
oxygen bond of 2.31 Å in length and an Mn 2+ – G 10.1 N 7 bond 2.24 Å in length
(see Table 6.4 ). If the A 9 prochiral R p oxygen is substituted by sulfur in a PS -
rescue experiment, Mg 2+ no longer catalyzes the cleavage reaction but catalysis
can be rescued by Cd 2+ addition.^31 P NMR shows that Cd 2+ does indeed bind
to the A 9 R p phosphorothioate. However, no ground - state crystallographic
structure shows the A 9 /G 10.1 site as being close to the cleavage site. This led
the Herschlag group to present their hypothesis that a single metal ion coor-
dinates to both the A 9 /G 10.1 and the cleavage site in the ribozyme – substrate
complex transition state.^45 This hypothesis was in turn challenged by the Scott
group showing by molecular dynamics calculations that the proposed single