The catalytic mechanism of dihydrofolate reductase is evaluated with Poisson-Boltzmann electrostatic and quantum chemical vibrational frequency calculations. The results indicate that an elevated pK(a) of 6.5 associated with the chemical step is due to the formation of the enol tautomer of the substrate’s pterin ring. The tautomer is induced to form as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the binding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization of the substrate, resulting in active site electrical neutrality and activation of the substrate for catalysis. These results require a reinterpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly protonated Quantum chemical vibrational frequency calculations demonstrate that the enol tautomer undergoes a Raman active vibrational perturbation at a frequency similar to that observed experimentally. Furthermore, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pK, for this residue being shifted from 2.6 in solution to below zero while bound to the protein. The conclusions of this work are independent of the protein dielectric constant in the range of 4-20.