276 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
Additional interactions and rearrangements in the transition state with
other rescuing bases may take place because it is known from crystal structures
that substrate atoms are not in line for nucleophilic attack in the hammerhead
ribozyme (at least not in the published crystal structures). Also, a metal ion
located∼ 20 Å away from the catalytic site was shown to be crucial for catalysis.
This same metal ion appeared likely to take on an additional ligand in the
transition state, suggesting that conformational changes had to take place
before catalysis.^38
Herschlag and co - workers continued to study the rescue of A9X using 28
different bases. Only purine bases effected rescue. Krescue was ∼ 30 - fold higher
upon adding adenine than for purine itself (see Figure 6.16 ), indicating that
the 6 - amino group of the base is important for rescue. Other experiments
indicated that adenine positions 2 and 3 are not important for transition state
interactions. Modifi cation of adenine to 1 - deazaadenine and 3 - deazaadenine
indicated that these positions do not engage in critical transition state interac-
tions either.
Changing A9X to A9 Φ (where Φ is a phenyl group, see Figure 6.14 )
decreased catalysis only a modest amount, indicating that stacking interactions
could be important for the A 9 position. Surprisingly, exogenous guanine
rescued catalysis better than exogeneous adenine, indicating that a hydrogen -
bonding pattern is more important than the individual base itself. The authors
indicate the possible hydrogen - bonding pattern for guanine to its pairing
partner G 12 in Figure 8 of reference 42. Overall, the A 9 position appeared to
serve a positioning or structural role in hammerhead catalysis. Similar behav-
ior was seen for the A13X variant hammerhead: pyrimidines do not cause
rescue but exogenous guanine does not cause rescue either. One purine, 2 -
methyladenine (Figure 6.16 ) does afford modest rescue, and the authors
believe that this may be due to fortuitous hydrophobic interactions within the
hammerhead central core. In the crystal structure (PDB: 1MME) the base G 12
Figure 6.16 Bases used in substitution and rescue experiments.N
NH NNPurine1
2
37
9NH NNNH H1-Deazaadenine7 6
91
3NN
HNNH H3-Deazaadenine7 6
91
3NNH NNNH HAdenine6 7 6
91
3NNH NNNH HCH 3
2-Methyladenine7 6
91
3NNH NNH H7-Deazaadenine7 6
91
3