The 9DB1 DNAzyme follows an addition-elimination (AN+DN) two-step mechanism, involving a phosphorane intermediate, where the 3'-hydroxyl group (nucleophile) of one RNA fragment attacks the 5'-triphosphate of another RNA fragment. This mechanism does not involve a divalent metal cation in agreement with the experimental evidence. The process is assisted by two proton transfers that activate the nucleophile (first step) and the leaving group (second step). The dA13 nucleotide is not directly involved in the reaction. However, it plays an important role in determining the regioselectivity of the process: since the dA13 phosphate forms a strong hydrogen bond with the 2'-hydroxyl, only the 3'-hydroxyl can behave as a nucleophile and form the new 3'-5' bond. In silico mutagenesis, where the dA13 phosphate oxygen involved in the hydrogen contact was replaced by a sulfur atom, causes a significant rearrangement of the A50 ribose position with an increase in the activation barrier and a consequent lower enzymatic activity in agreement with the experimental evidence. A similar effect is determined by the replacement of the 2'-hydroxyl with different groups such as F, H, and OMe.