268 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
coordination of a metal cation to the A 9 – G 10.1 Me 2+ binding site appeared to
be through the pro - S p oxygen rather than the pro - R p oxygen of A 9 ’ s phos-
phate); (3) the crystals were grown in a much lower concentration of salt; and
(4) the crystal packing scheme was very different. In spite of these differences,
the overall structure appeared similar. Magnesium ions were soaked into the
PDB: 1MME hammerhead crystal allowing the identifi cation of fi ve Mg 2+ -
binding sites, including one near the ribozyme – substrate complex catalytic
pocket. In helix I a Mg 2+ interacts with N7 and O6 of both G2.2 and G2.3.
Another Mg 2+ is near the cleavage site, in the CUGA loop, where it interacts
with several bases, including A 1.1, C 3 , U 4 , U 7 , G 8 , and C 17. This magnesium ion,
close to the cleavage site, may have a catalytic role. Another two sites were
observed in domain II. At one of these, the Mg 2+ interacts primarily with the
pro - R - oxygen of the A 9 phosphate and N7 of G 10.1. Neither X - ray structure
(PDB: 1HMH nor 1MME) showed the proper inline positioning of the scissile
phosphate group for the S N 2 cleavage mechanism to take place.
Other researchers attempted to clarify the differences reported between
the 1HMH and 1MME hammerhead ribozyme structures. Herschlag ’ s group
fi rst traced the history of biochemical data accumulated for the hammerhead
ribozyme and then continued with their own experiments. It had been shown
previously in phosphorothioate interference experiments that the pro - R p
oxygen of A 9 ’ s phosphate was important for hammerhead substrate cleavage.^36
Nucleotide substitution experiments had previously implicated G 10.1 as impor-
tant for ribozyme catalytic activity.^37 In their 1997 Journal of Biological Chem-
istry article, the Herschlag group substituted sulfur into the pro - R p and pro - S p
phosphate oxygen positions at A 9 and then followed catalytic activity in the
absence and presence of the thiophilic, soft Cd 2+ ion.^38 The experiments were
conducted on the HH α 1 (Figure 6.12 ) and HH16 (Figure 6.11 ) hammerhead
ribozyme constructs, constructs that differ in some residue positions but that
have the same set of conserved residues as those used to determine the crystal
structures PDB: 1HMH and 1MME (the RNA 6 construct shown in Figure
6.10 ). Both the HH α 1 and HH16 ribozymes showed loss of catalytic activity
in the A 9 pro - R p - phosphorothioate in the presence of Mg 2+ that could be
rescued by addition of 100 μ M Cd 2+. These results indicated that a metal ion
coordinated at the pro - R p oxygen of position 9 is critical for effi cient catalysis.
By studying the concentration dependence of Cd 2+ with respect to observed
rates of the catalytic reaction, the researchers could calculate an apparent dis-
sociation constant of KdCd= 25 μM in the ground state. Using transition state
theory and the equation
KK
k
k
ddCd Cd
Cd∓
= ()^2
2(6.1)
the researchers were able to calculate an apparent dissociation constant of
KdCd‡=25.nM in the transition state. This much stronger Cd 2+ binding in the
transition state was interpreted to indicate at least one additional ligand