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

Abstract

The RNA-cleaving 8-17 DNAzyme, which is a metalloenzyme that depends on divalent metal ions for function, is the most studied catalytic DNA in terms of its mechanism. By the end...

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

“…This analysis can also help to evaluate the influence of the monovalent cation over the pK a of the hydrated divalent metal, identified experimentally as a general acid in the mechanism of catalysis. 18 To achieve this goal, we performed activity assays with the variant 8-17 G14-AP, in which G14 has been replaced by a 2-aminopurine, which has a lower N1 pK a (pK a,exp,Mg = 5.3 and pK a,exp,Pb = 5.4). 17,18 We measured the RNA-cleavage reaction rate as a function of pH, in the presence of 100 µM Pb 2+ with 25 mM Na + and we compared these results with the pH-rate profile at 100 mM Na + , measured previously by our group.…”

“…18 To achieve this goal, we performed activity assays with the variant 8-17 G14-AP, in which G14 has been replaced by a 2-aminopurine, which has a lower N1 pK a (pK a,exp,Mg = 5.3 and pK a,exp,Pb = 5.4). 17,18 We measured the RNA-cleavage reaction rate as a function of pH, in the presence of 100 µM Pb 2+ with 25 mM Na + and we compared these results with the pH-rate profile at 100 mM Na + , measured previously by our group. 18 Fig.…”

“…17,18 We measured the RNA-cleavage reaction rate as a function of pH, in the presence of 100 µM Pb 2+ with 25 mM Na + and we compared these results with the pH-rate profile at 100 mM Na + , measured previously by our group. 18 Fig. 4 shows the pH-rate profiles fitted to a model for general acid-base catalysis (eqn (2)).…”

“…5A). 11,12,17,18 The combination of these two catalytic strategies, besides the in-line fitness (α-catalysis) exposed in the crystal structure, would explain the rate of enhancement achieved in the RNAcleaving reaction catalyzed by the 8-17 DNAzyme under optimal conditions. A similar combination of strategies has been suggested as a part of the mechanism of the pistol ribozyme.…”

“…On the other hand, the pH-rate profile of an 8-17 DNAzyme mutant fits well into a projected model where the divalent metal ion acts as a general acid for both cofactors (comparing Pb 2+ and Mg 2+ ), despite the divergent folding pattern found. 12,18 Therefore, what are the fundamental differences in the reaction mechanism of the 8-17 DNAzyme depending on the nature of the divalent metal ion used as the cofactor?…”