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be experimentally observable in the unmodified ribozyme), then identical
profiles will be observed for both cleavage and ligation reactions.
In fact, replacing G8 with nucleobases of lower p
K
a
provides good evi-
dence for participation of the nucleobase at this position, and thus for general
acid-base catalysis by two nucleobases. Substitution of G8 by diaminopurine
or 2-aminopurine,
80
imidazole,
85
or 8-azapurine
90
resulted in cleavage
becoming slower at higher pH, consistent with general acid-base catalysis
involving two nucleobases.
We have extensively analyzed the available evidence for the catalytic
mechanism of the hairpin ribozyme and do not propose to repeat these argu-
ments here.
89
We conclude that the available data are consistent with general
acid-base catalysis by A38 and G8, and that there are no data that conflict
with this mechanism. Recent new data further support this.
91
We have
shown that the large reduction in cleavage rate on substitution of A38 by
purine (A38P) can be reversed by replacement of the 5
0
-oxygen atom at
the scissile phosphate by sulfur. This is consistent with A38 acting as general
acid in the unmodified ribozyme, but would not be expected if it were only
stabilizing the transition state by hydrogen bonding or electrostatic interac-
tion. The cleavage rate by the A38P ribozyme of the 5
0
-PS substrate was
found to increase in a log-linear manner with pH, indicative of a require-
ment for a deprotonated base with a relatively high p
K
a
. Moreover, when
G8 was substituted by diaminopurine, the 5
0
-PS substrate cleavage rate
increased with pH to
6, above which the rate stayed constant. The appar-
ent p
K
a
was consistent with this nucleotide acting in general base catalysis.
We conclude that only general acid-base catalysis by A38 and G8 is con-
sistent with the experimental data. Despite the very different overall archi-
tectures of the hairpin and VS ribozymes, it seems that their active sites and
catalytic mechanisms are likely to be quite similar with key components hav-
ing the same functions (i.e., the adenine and guanine nucleobases acting in
the cleavage reaction as the general acid and general base, respectively).
3. GENERAL THEMES
It is clear that there is a strong mechanistic similarity between the
hairpin and VS ribozymes, and that nucleobase-mediated general acid-base
catalysis makes a major contribution to the rate enhancements of both
ribozymes. Both employ a guanine as general base and adenine as general
acid in cleavage, and the active sites of the ribozymes are very similar. This
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