The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation - Progress in Molecular Biology and Translational Science - page 6

performing experimental controls for time-resolved crystallographic freeze-
trapping experiments in crystals of the minimal hammerhead ribozyme. 36-38
It now appears that RNA folding and nonspecific electrostatic transition-
state stabilization accounts for much, if not all, of the catalytic enhancement
over background rates found with these ribozymes. 36,37 For example, ham-
merhead 16.1, which is considered to be an optimized hammerhead ribo-
zyme sequence for single-turnover reactions, cleaves only threefold faster
in the presence of 10 mM MgCl 2 and 2 M Li 2 SO 4 than it does in the pres-
ence of 2 M Li 2 SO 4 alone. 36,37 The rates of hairpin and VS ribozymes in
2MLi 2 SO 4 actually exceed those measured under “standard” low ionic
strength conditions, and the rate of cleavage for the non-optimized ham-
merhead sequence used for crystallization is fivefold enhanced in 2 M
Li 2 SO 4 alone versus standard reaction conditions. The non-optimized
sequence used for crystallization tends to form alternative, inactive structures
in solution, such as a dimer of the enzyme strands, which dominate at lower
ionic strength.
This result implied that any chemical role of Mg 2 þ ion in the ribozyme
reaction was likely to be one of comparatively nonspecific electrostatic
stabilization rather than more direct participation in the chemical step of
catalysis. Moreover, the result implied that the RNA itself was an active par-
ticipant in the chemistry of catalysis rather than serving as a passive scaffold
for binding metal ions that served the roles of general acid and base catalysts.
With the subsequent structural elucidation of the hairpin 39,40 and full-length
hammerhead 41 structures, it was, in fact, revealed that RNA bases and other
functional groups were positioned to provide the moieties likely responsible
for acid-base catalysis.
2.1.3 Acid - base chemistry
Originally, hydrated Mg 2 þ and other hydrated divalent metal ions were
thought to play the direct chemical role of general base and general acid
in ribozyme catalysis, with the RNA itself serving as an ancillary and passive
scaffold upon which metal ions would bind and would be positioned in the
active site.
With the discovery that the hairpin, hammerhead, and VS ribozymes
were not strictly metalloenzymes, 36,37 it became apparent that in at least these
three cases, the RNA itself must be an active participant in the chemistry of
catalysis rather than serving merely as a metal ion-binding scaffold. The crys-
tal structure of the hairpin ribozyme, 40 in contrast to the HDV ribozyme 42,43
that is in fact a metalloenzyme, soon validated this prediction. However, it