Ab initio and density functional calculations have been carried out to more fully understand the factors controlling the catalytic activity of the Thermus aquaticus DNA methyltransferase (MTaqI) in the N-methylation at the N(6) of an adenine nucleobase. The noncatalyzed reaction was modeled as a methyl transfer from trimethylsulfonium to the N(6) of adenine. Activation barriers of 32.0 kcal/mol and 24.0 kcal/mol were predicted for the noncatalyzed reaction in the gas phase by MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. Calculations performed to evaluate the effect of substrate positioning in the active site of MTaqI on the reaction determine the barrier to be 23.4 kcal/mol and 17.3 kcal/mol for the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) gas phase calculations, respectively. The effect of hydrogen bonding between the N(6) of adenine and the terminal oxygen of Asn-105 on the activation barrier was also studied. A formamide molecule was modeled into the system to mimic the function of active site residue Asn-105. The activation barrier for this reaction was found to be 21.8 kcal/mol and 15.8 kcal/mol as determined from the MP2/6-31+G(d,p)//HF/6-31+G(d,p) and B3LYP/6-31+G(d,p) calculations, respectively. This result predicts a contribution of less than 2 kcal/mol to the lowering of the activation barrier from amide hydrogen bonding between formamide and N(6) of adenine. Comparison of the reaction coordinates suggest that it is not the hydrogen bonding of the Asn-105 that lends to the catalytic prowess of the enzyme since the organization of the substrates in the active site of the enzyme has a far greater effect on reducing the activation barrier. The results also suggest a stepwise mechanism for the removal of the hydrogen from the N(6) of adenine as opposed to a concerted reaction in which a proton is abstracted simultaneously with the transfer of the methyl group. The hydrogen on the N(6) of the intermediate methyl adenine product is far more acidic than in the reactant complex and may be subsequently abstracted by basic groups in the active site that are too weak to abstract the proton before the full sp(3) hybridization of the attacking nitrogen.