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cleavage rate pH profile, with an upper p
K
a
reduced by 0.3 units,
22
where
the expected p
K
a
of inosine is
0.5 unit lower than that of guanine. Inter-
estingly, the lower p
K
a
was also reduced by 0.4 unit, suggesting that A756 is
close enough to G638 to be influenced by the substitution. When our C
2
-
linked imidazole nucleoside
60
was placed at this position, we obtained an
upper p
K
a
of 7.0 (unpublished data). Substitution of G638 by 2,6-
diaminopurine (DAP) with a normal p
K
a
of 5.1 produced a rate of cleavage
that was reduced 1000-fold relative to the natural sequence at pH 8. How-
ever, it significantly restored activity at lower pH with a corresponding p
K
a
of 5.6. The resulting rate was log-linear with pH over the range from 6 to
8 with a unit gradient. This indicates that general acid-base catalysis contrib-
utes over two orders of magnitude to the catalytic power of the VS
ribozyme.
The pH dependence of a reaction may reflect a change in the rate-
limiting step rather than the protonation state of reactants, but several lines
of evidence suggest that this is not the case for the VS ribozyme. Kinetic
isotope effects in a fast,
cis
-acting form of the VS ribozyme show that proton
transfer occurs in the transition state of the cleavage reaction.
62
In the
trans
form of the ribozyme, the central conversion of substrate to product is rate
limiting, with rapid and pH-independent substrate binding.
22
Finally, the
correlation between the p
K
a
of the nucleobase at position 638 and the
observed p
K
a
of the cleavage reaction strongly suggests that the rate of reac-
tion depends on protonation state of the nucleobase.
While a substantial part of the VS ribozyme rate enhancement may result
from general acid-base catalysis by A756 and G638, the pH profiles do not
allow us to determine which nucleobase is the acid, and which the base. The
two alternatives lead to identical predicted pH dependence. A similar
ambiguity existed for the HDV ribozyme, but was resolved by the use of
a5
0
-phosphorothiolate (5
0
-PS) substitution at the scissile phosphate.
63
The 5
0
-S atom is a much better leaving group than the normal O, thus
not requiring protonation. As a consequence, substitutions in the ribozyme
that impair the function of the general acid and lower the activity on the oxy
(5
0
-PO) substrate should have little effect on cleavage of a 5
0
-PS-containing
substrate. In other words, the 5
0
-PS substitution should “rescue” the cleav-
age activity of ribozyme in which the general acid has been removed. On the
other hand, impairment due to changes in the general base will not be res-
cued by 5
0
-PS substitution. In an analogous experiment, it was previously
shown that the cleavage of uridine 3
0
-(
p
-nitrophenyl phosphate) was unaf-
fected by an H119A mutation in ribonuclease A
64
because the
p
-nitrophenyl
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