The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a |
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Authors: | Smith M Duane Mehdizadeh Reza Olive Joan E Collins Richard A |
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Affiliation: | Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8. |
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Abstract: | Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pKa values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pKa values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k1, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pKa. The inference that k1 is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pKa is that of the general acid and the higher pKa is that of the general base. |
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Keywords: | ribozyme mechanism metal ions general acid–base catalysis pKa kinetic ambiguity |
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