A Mechanistic Comparison of the Varkud Satellite and Hairpin Ribozymes - Progress in Molecular Biology and Translational Science - page 113

does not exclude additional contributions to the total observed rate
enhancement. Stabilization by enhanced coordination of the transition state
was observed in crystal structures of the hairpin ribozyme 38 and may also
contribute to the VS ribozyme since the exocyclic amines of A756 and
G638 are necessary for full activity, 21,22 although electrostatic effects may
also contribute. However, the pH dependence of a G638DAP-substituted
VS ribozyme suggested that proton transfer contributes at least 100-1000-
fold to the catalytic power of the ribozyme. 22 General acid-base catalysis
is

therefore likely to be a very significant source of catalytic rate

enhancement.
General acid-base catalysis is not restricted to the hairpin and VS
ribozymes, but seems to be the major contributor to the rate enhancement
for all the nucleolytic ribozymes. In all cases, nucleobases seem to act as direct
participants and guanine is the most commonly used. Guanine nucleobases
appear to be strong contenders for the role of general base in cleavage reactions
catalyzed by the hammerhead ribozyme (G12) 92,93 and GlmS ribozyme (G33
or G40 depending on the organism). 94-96 However, the guanine and adenine
combination is only observed in the hairpin and VS ribozymes. The ham-
merhead ribozyme is proposed to employ a 2 0 -hydroxyl, 97 and the GlmS
ribozyme uses an exogenous glucosamine-6-phosphate as general acid in
cleavage reactions, 94,98 while the HDV ribozyme uses a Mg 2 þ ion to activate
the nucleophile, either as general base or possibly as a Lewis acid, 99,100 and a
cytosine nucleobase as general acid. 63,100-102 Thus it seems probable that gen-
eral acid-base catalysis is a source of catalytic rate enhancement in all the
nucleolytic ribozymes.
By contrast, the self-splicing intron ribozymes 103-106 and ribonuclease
P 107,108 have adopted a different strategy. They act as metalloenzymes that
use bound metal ions to activate, orient, and stabilize catalytic components.
As we have pointed out earlier, RNA is essentially recapitulating the two
alternative strategies of protein enzymes that carry out phosphoryl transfer
reactions exemplified by ribonuclease A (using two histidine side chains
in general acid-base catalysis) 109 and many other nucleases and polymerases
that use a two-metal ion mechanism. 110 As a polyelectrolyte, it is not diffi-
cult for RNA to create selective metal ion binding pockets. Thus it seems
probable that the metalloenzyme mechanism is likely to have arisen before
general acid-base catalysis in the chemical evolution of life, and clearly the
nucleobases suffer a significant disadvantage having p K a values far from neu-
trality. Nevertheless, the nucleolytic ribozymes are as efficient as they need
to be in their contemporary biological roles.