Probing Metal-Dependent Phosphate Binding for the Catalysis of the 17E DNAzyme.

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb ²⁺ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni ²⁺ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, R _p and S _p , indicated that Mg ²⁺ , Mn ²⁺ , Fe ²⁺ , and Co ²⁺ had the highest S _p : R _p activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb ²⁺ , suggesting inner-sphere coordination. The pH-rate profiles showed that Pb ²⁺ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent p K _a and the p K _a of metal-bound water. Combining previous reports and our current results, we propose that Pb ²⁺ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.