HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 130

ribozyme failed due to spectral heterogeneity. This result led to the conclusion
that a protonated C75 is likely associated with the phosphorane intermediate
(or transition state) of the reaction, thus supporting a general acid or an elec-
trostatic role for the nucleobase. Similar results were obtained by measuring
the fluorescence of aminopurine placed next to the active-site cytosine. 77
4.2. Metal ion requirements
A variety of divalent metal ions accelerate the ribozyme cleavage reaction.
Self-scission is observed upon incubation with Mg 2 þ ,Ca 2 þ ,Sr 2 þ ,Ba 2 þ ,
Mn 2 þ ,Co 2 þ , and to a lesser extent Cd 2 þ and Ni 2 þ ions, but not in the pres-
ence of cobalt(III) hexammine, an exchange-inert structural analog of
hexahydrated divalent metal ions. 2,10,12,27,35,51,66,78,79 Self-scission is also
observed at high concentrations of monovalent ions, but the rate constants
are more than two orders of magnitude lower than in the presence of
divalent metal ions. 46,73 These observations suggest that divalent metal ions
promote catalysis, but the lack of metal ion preference coupled with
cobalt hexamine inhibition suggests that a hydrated form of divalent
metal ion is required for efficient self-cleavage. Sulfur substitution of
bridging and nonbridging oxygens on the scissile phosphate does not
switch the metal ion preference toward thiophilic metal ions, suggesting that
these positions are not directly contacted by the metal ions acting as
Lewis acids. 80 These observations cannot rule out inner sphere coordination
by a metal ion to other active-site groups. However, such coordination
would require the active site to be able to accommodate multiple different
metal ions.
Metal ion accommodation by the active site is further supported by
experiments showing that changing the scissile phosphodiester configura-
tion from 3 0 -5 0 to 2 0 -5 0 resulted in a change of preference from Ca 2 þ to
Mg 2 þ . 66 While the 3 0 -5 0 phosphodiester is cleaved somewhat faster in
Ca 2 þ , the 2 0 -5 0 linkage is cleaved two orders of magnitude slower in
Mg 2 þ , and in the presence of Ca 2 þ the rate drops to background levels.
Interestingly, the apparent p K a values of the reaction in Mg 2 þ are indepen-
dent of the type of phosphodiester undergoing scission.
Analysis of the divalent metal ion dependence in the presence of 1 M
NaCl revealed that the genomic HDV ribozyme binds a structural Mg 2 þ
with higher affinity than the active-site Mg 2 þ . 72 The active-site metal ion
appears to act on the 2 0 nucleophile because the apparent K D of Mg 2 þ does
not change between C76 and C76U variants when the 5 0 leaving group is