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respectively, can be summarized as follows: (1) in all crystal structures, the
nucleobase is located within several
˚
ngstroms of the scissile phosphate
34-36
;
(2) the C75/76U and C75/76G variants do not support catalysis and C75/
76A shows only partial activity
45,46
; (3) the kinetic pH profile of the C75/
76A variant shows an apparent p
K
a
shifted by a number of pH units
corresponding to the difference between the p
K
a
values of free cytidine
and adenosine
46
; (4) substitution of the active-site cytosine with cytosine
analogs similarly shifts the apparent kinetic p
K
a
by approximately the same
amount as the p
K
a
difference between the free bases
67,68
; (5) self-scission of
the inactive C75/76U or deleted C75/76 at this position can be rescued by
exogenous nitrogenous bases including free cytosine, imidazole, and other
analogs with the kinetic p
K
a
values tracking the p
K
a
values of the free
bases
45,69,70
; and (6) proton inventory experiments and solvent kinetic iso-
tope effects of the C75, C75A, and imidazole-rescued inactive ribozymes
support a proton transfer from this position in the ribozyme (
Fig. 4.3
).
46,70,71
For the majority of studies conducted on the HDV ribozymes, the
active-site cytosine could be assigned a proton-transferring role as either
an acid or a base. However, three experiments strongly implied that the
nucleobase acts as a general acid in the mechanism. First, a sulfur substitution
of the 5
0
bridging oxygen of G1 produced an activated leaving group that no
longer required the participation of the nucleobase in catalysis.
67
Second, a
crystal structure of an inhibited ribozyme located the C75 residue within
hydrogen bonding distance of the leaving group where it would act as a gen-
eral base, as would be the case with the C75U mutant, rather than in prox-
imity to the 2
0
OH nucleophile.
35,36
Third, the rate of self-scission in the
absence of Mg
2
þ
results in a pH profile and a kinetic solvent-isotope effect
that support transfer from a protonated cytosine.
46,49,71-73
Kinetic pH profiles, proton inventory experiments, and Brønsted anal-
ysis indicated that at least one proton is transferred during the rate-limiting
step of the reaction, most likely the one from a protonated C75/76 to the
oxyanion leaving group of G1.
69,71,74
In order for the cytosine to efficiently
transfer a proton to the 5
0
oxyanion leaving group, its p
K
a
needs to be shifted
from approximately 4.2 toward neutrality. Several biophysical experiments
have been designed to measure the equilibrium p
K
a
of the active-site cyto-
sine in various ribozyme constructs. Raman crystallography detected the
protonation state of inhibited precursor forms of the ribozyme (2
0
-deoxy,
2
0
-methoxy, or 2
0
-fluoro substitutions at the
1 position) and yielded a
p
K
a
of 6.1-6.2 and 6.4 in 20 and 2 mM Mg
2
þ
, respectively, for the
2
0
-methoxy inhibited ribozyme.
75
The RNA construct used was similar
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