300 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
for the S N 2 mechanism. However, they maintain that it is still unclear whether
these changes are on the actual hammerhead reaction pathway. For instance,
most of the changes in the crystal structures appear near (proximal to) the
cleavage site, whereas few changes are observed for essential functional groups
in domain II distal to the cleavage site. The reviewers suggest that the Her-
schlag group metal ion rescue experiments^45 may be correct in stating that the
same Mg 2+ coordinates to the A 9 /G 10.1 and the scissile phosphate in the transi-
tion state even though these sites are 20 Å apart in the crystal structures (see
Figure 9 of reference 61 ). Metal ion rescue experiments and the one - metal
theory have already been discussed in relation to suggestions by the Scott
group that conformational changes to this transition state are energetically too
costly.^49 In support of the Herschlag hypothesis, hydroxyl radical footprinting
experiments suggest a more compact and less solvent - exposed conformation
for the hammerhead in solution compared to conformations found in the
crystal structures.^56
Blount and Uhlenbeck believe that answers may be found to the above -
described inconsistencies for the hammerhead ribozyme by studying naturally
occurring hammerheads in which the closing loops of stems I and II interact
with each other to produce a much more compact and closely packed ham-
merhead active conformation.^63 The tertiary interaction between the stems
appears to lower the Mg 2+ concentration required for full catalytic activity. The
reference 61 and 63 authors believe that further study of the naturally occur-
ring hammerheads that exhibit close conformational interactions between
domains I and II may lead to a more unifying view of the catalytic cleavage
mechanism.
In 2006, the 2.2 - Å resolution crystal structure of a full - length Schistosoma
mansoni hammerhead ribozyme was determined by Martick and Scott.^64
(PDB: 2GOZ). The researchers crystallized a full - length hammerhead ribo-
zyme of 43 nucleotides (nt) in complex with a 20 - nt substrate. No metal ions
are present in the X - ray crystallographic structure. The full - length hammer-
head ribozyme contained the rate - enhancing peripheral domain and had a
catalytic core that was very different from the catalytic core present in the
structure of the “ minimal ” hammerhead constructs that have been discussed
here — that is, all the previously discussed hammerhead constructs have lacked
the peripheral domain present in PDB: 1GOZ.^65 The X - ray structure revealed
how tertiary interactions that occur in regions remote from the active site
prime the ribozyme for catalysis. For instance, nucleotides G 12 and G 8 are
positioned in PDB: 1GOZ to carry out their previously suggested roles as
acid – base catalysts. The nucleophile is aligned for an S N 2 reaction with a
scissile phosphate positioned proximal to the A 9 phosphate. Previously
unexplained roles of other conserved nucleotides became apparent when
observing the distinctly new fold in the PDB: 1GOZ structure. The newly
observed ribozyme – substrate interactions allowed the reference 64 authors to
explain many of the previously confl icting experimental results. Interested
readers should consult reference 64 for more detail.