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showed the amine group positioned at the site of cleavage.
12-15,17
Specifi-
cally, it has been proposed that the coenzyme amine acts as a general acid,
protonating the 5
0
-oxygen leaving group of G1 during
glmS
ribozyme self-
cleavage.
12,13
Molecular dynamics simulations have also predicted that
GlcN6P acts as a general acid based on the results that upon binding of
GlcN6P to
glmS
ribozyme, the p
K
a
of the amine functionality decreased
by 2.2 pH units from the solution p
K
a
of 8.2 due to the active site environ-
ment. In contrast, the p
K
a
of the amine group slightly increased to about
8.4 upon binding to a G40A inactive mutant of the
glmS
ribozyme
(
Thermoanaerobacter tengcongensis
ribozyme numbering corresponding to
G33 in the
B. cereus
ribozyme).
38
The hypothesis that the coenzyme amine
acts as a general acid predicts that the N1 of G40 functions as the general
base. Although there is some support for this hypothesis, there is much con-
flicting evidence.
10. pH-REACTIVITY PROFILES PROVIDE MECHANISTIC
INSIGHT
A mechanistic interpretation of the pH-reactivity profile for the
glmS
ribozyme has been previously proffered and provides insight into how the
catalyst might function.
42
A similar type of analysis had previously been per-
formed for the HDV ribozyme.
43,44
To summarize, the
glmS
ribozyme
exhibits a pH-reactivity profile with an apparent p
K
a
above which a slope
of zero may reflect the inverse relationship of the catalyst's general acid
and general base contributions. A number of pH-reactivity profiles for
the
glmS
ribozyme have been performed that all reveal a reduced apparent
reaction p
K
a
of 6.9-7.8 relative to the p
K
a
of 8.2 for GlcN6P in solu-
tion.
6,16,36,39
By analogy to the HDV ribozyme, if GlcN6P functions pri-
marily as a general acid catalyst for
glmS
ribozyme self-cleavage following
its solution p
K
a
of 8.2, there exists a requirement for a relatively strong gen-
eral base catalyst with a p
K
a
of
10 to achieve the observed kinetic profile
(
Fig. 5.3
). Crystallographic studies proposed that the N1 of G33 functions as
the general base due to its position adjacent to the 2
0
-OH of A-1 at the scis-
sile phosphodiester linkage,
12,13
and the p
K
a
of this nucleobase functionality
is consistent with this model. However, a number of observations contradict
this relatively simple interpretation of the pH-reactivity profile. Primarily,
the model predicts that such an independently functioning general base cat-
alyst would support measurable activity in the absence of GlcN6P as a gen-
eral acid catalyst at neutral or basic pH. However, the ribozyme is inactive in
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