DHFR's enzymatic mechanism is shown to be pH dependent, particularly the hydride transfer step, since pH changes are shown to have remarkable influence on the electrostatics of the active site and the ionization state of its residues. The acidity of the targeted nitrogen on the substrate is important in the binding of the substrate to the enzyme's binding site which is proved to be hydrophobic even though it has direct contact to water. Asp27 is the only charged hydrophilic residue in the binding site, and neutralization of the charge on Asp27 may alter the pKa of the enzyme. Asp27 plays a critical role in the catalytic mechanism by helping with protonation of the substrate and restraining the substrate in the conformation favorable for the hydride transfer. The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation. A water molecule is proved to be involved in the protonation step. Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.