The role of Na+ in catalysis by the 8-17 DNAzyme.

The 8-17 DNAzyme is the most studied deoxyribozyme in terms of its molecular mechanism; hence it has become a model system to understand the basis behind DNA catalysis. New functional studies and the recent attainment of high-resolution X-ray structures, in addition to theoretical calculations have offered a great opportunity to gain a broader comprehension of its mechanism; however many aspects are unclear yet, especially regarding the precise role of metal ions in catalysis. Recently, molecular dynamics simulations have suggested for the first time a specific and dynamical participation of Na+ in the mechanism through the reaction pathway, besides the roles proposed for divalent metal cofactors. Herein, we present experimental evidence of a cooperative role of the monovalent cation Na+ in catalysis that is in line with these theoretical suggestions. Our findings show a clear influence of the concentration of Na+ on the activity of the 8-17 DNAzyme when Pb2+ is used as the cofactor. Interestingly, this effect is not noticed with Mg2+, indicating a particular contribution of the monovalent ion to catalysis that would operate preferentially with Pb2+. We have also found that Na+ affects the pKa of the general base and the general acid, indicating its influence on general acid-base catalysis, already identified as part of the mechanism of the 8-17 DNAzyme. Finally, our results emphasize the need to consider Na+ carefully in the design and analysis of functional studies of catalytic DNAs and its possible specific role in their mechanisms.

Hydrated metal ion as a general acid in the catalytic mechanism of the 8-17 DNAzyme.

The RNA-cleaving 8-17 DNAzyme, which is a metalloenzyme that depends on divalent metal ions for its function, is the most studied catalytic DNA in terms of its mechanism. By the end of 2017, a report of the crystal structure of the enzyme-substrate complex in the presence of Pb2+ probed some of the previous findings and opened new questions, especially around the participation of the metal ion in the catalytic mechanism and the promiscuity exhibited by the enzyme in terms of the metal cofactor required for catalysis. In this article we explore the role of the divalent metal ion in the mechanism of the 8-17 DNAzyme as a general acid, by measuring the influence of pH over the activity of a slower variant of the enzyme in the presence of Pb2+. We replaced G14, which has been identified as a general base in the mechanism of the enzyme, by the unnatural analog 2-aminopurine, with a lower pKa value of the N1 group. With this approach, we obtained a bell-shaped pH-rate profile with experimental pKa values of 5.4 and 7.0. Comparing these results with previous pH-rate profiles in the presence of Mg2+, our findings suggest the stabilization of the 5'-O leaving group by the hydrated metal ion acting as a general acid, in addition to the activation of the 2'-OH nucleophile by the general base G14.