A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes - Progress in Molecular Biology and Translational Science - page 106

All the available data are therefore consistent with a catalytic mechanism
for the VS ribozyme cleavage reaction in which G638 acts as general base to
deprotonate the 2 0 -O nucleophile, and A756 is the general acid protonating
the 5 0 -oxyanion leaving group. By the principle of microscopic reversibility,
in the ligation reaction a protonated G638 should act as the general acid pro-
tonating the 2 0 -oxyanion leaving group, and an unprotonated A756 as gen-
eral base deprotonating the 5 0 -O nucleophile that attacks the cyclic

phosphate.
Having assigned the acid and the base, it is interesting to compare the VS
ribozyme to its protein equivalent RNase A. Given p K a values of 5.2 and 8.4
for the ribozyme, the product f A 10 4 and

thus k cat is 330 s 1 under the conditions of the assay. This is not much lower
than the k cat for RNase acting on various substrates, of

f B achieves a maximum of 3.8

1400 s 1 . 66 Of
course, the superiority of the protein enzyme lies in the maximum f A f B

of 0.25.
Is this the complete story of how the VS ribozyme reaction is catalyzed,
or could other components to the overall rate enhancement exist? The direct
participation of a site-bound metal ion, either as a Lewis acid or in general
acid-base catalysis, is unlikely because the ribozyme is active in monovalent
ions. 67 However, some electrostatic stabilization of the transition state by
nonspecifically bound ions may occur. It is possible that the transition state
might also be stabilized by interactions between the key nucleobases and the
phosphorane, as suggested by the crystal structure of the hairpin ribozyme
(see below). 38 Alternatively, a protonated adenine (other than A756) might
stabilize the dianionic phosphorane, as proposed for the hairpin ribozyme. 68
However, these effects seem unlikely to be major contributors to the ribo-
zyme rate enhancement because they should not be reversed by activation of
the leaving group with 5 0 -PS substitution. If the sole function of the ribo-
zyme is to present A756 so that it can transfer a proton in the transition state,
then the ribozyme might be unnecessary for the cleavage of the 5 0 -PS sub-
strate. While the cleavage rate of the natural sequence (i.e., G638
unmodified) 5 0 -PS substrate in the absence of ribozyme was found to be

750-fold faster than the oxy form, the rate was nevertheless increased
an additional 70-fold by the addition of a trans -acting ribozyme. 65 Interac-
tion of the substrate with the ribozyme probably remodels the structure of
the substrate into a conformation required for full activity. This might bring
the nucleobase of G638 closer to the 2 0 -OH group of G620. This would also
be anticipated to reposition the 2 0 -O for in-line nucleophilic attack, which
should generate a further modest rate enhancement.