284 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
hammerhead – metal ion interactions that stimulate catalytic rate in the pres-
ence of divalent ions could be absent with monovalent ions.
To test this hypothesis, the Herschlag group conducted the phosphorothio-
ate replacement and rescue experiments as described in the reference 45
article discussed above. The previous result indicated that if the P9 pro - R p
phosphoryl oxygen and that of P1.1 in the cleavage site were replaced by
sulfur, cleavage rates in Mg 2+ were greatly decreased (by 10^3 - to 10^4 - fold).
Using the same phosphoryl oxygen – sulfur replacements, the reference 47
authors predicted that if the monovalent Li + ion were not used to coordinate
the P9 and P1.1 pro - R p phosphoryl oxygen sites, the amount of rate decrease
would be considerably less. Indeed a rate decrease of approximately 10 - fold
was found for the reactions in 4 M Li + substantiating this hypothesis. This and
other experimental results led the researchers to conclude that the substantial
difference between catalytic stimulation by monovalent and divalent ions is
found at the P9/P1.1 site. Overall conclusions from this work and other obser-
vations concerning the metal ion ’ s role in structure versus its role in catalysis
were as follows: (1) Hammerhead cleavage reactions in the presence of mon-
ovalent or divalent metal ions are very similar; (2) the role of the metal or
even the ammonium ion (NH 4 + ), also found to increase hammerhead catalytic
rate, may be to neutralize phosphate repulsion and facilitate hammerhead core
folding into the catalytic conformation; (3) some part of hammerhead rate
enhancement does involve at least one divalent metal ion; and (4) the divalent
ion may provide its catalytic contribution by electrostatic stabilization of the
transition state in addition to its role in facilitating formation of the active
hammerhead structure.
The hypothesis that one divalent metal ion coordinated to both the A 9 /G 10.1
and the scissile phosphate at P 1.1 in a hammerhead catalytically active transi-
tion state was challenged by Murray and Scott in aJournal of Molecular
Biology paper published in 2000.^49 The authors initially constructed models
using the graphics display program O.^50 The structures were then refi ned in
X - PLOR 3.8.^51 Their goal was to use molecular modeling to defi ne a structure
that would obey bonding, RNA stereochemistry, and the inline S N 2 mechanism
for the phosphodiester bond cleavage, as well as incorporate the Herschlag
group divalent - ion - coordinating hypothesis. As described above, the Herschlag
group proposed that to achieve the catalytically active transition state with
one coordinating divalent metal ion, the stem II helix and neighboring domain
II with the A 9 /G 10.1 metal ion coordination site remain nearly unchanged
and that the domain I (uridine turn) region and its associated cleavage site
residue, C 17 , changes conformation from the ground - state crystal structure to
accommodate metal ion binding to a P 1.1 pro - R p phosphate oxygen.^49 In refer-
ence 49 , the starting point for all molecular modeling was the authors ’ best
catalytically active hammerhead crystal structure with Mn 2+ bound (PDB:
300D). Murray and Scott found that only one Mg 2+ coordinating position (in
its octahedral coordination sphere) could accommodate the single divalent ion
hypothesis. This position allowed a stereochemically plausible C 17 – U 16.1 phos-