Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

Dihydrofolate reductase is a classic drug target because it promotes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to yield 5,6,7,8-tetrahydrofolate (THF), which is involved in the biosynthesis of purines, thymidylate, and several amino acids. It is also a popular model system for various biochemical/biophysical studies. However, there are many unresolved mechanistic issues regarding the mechanism of catalysis. We combined primary, solvent, and multiple kinetic isotope effects; their temperature dependence; theoretical calculations; and site-specific mutagenesis to elucidate the reaction mechanism, which involves stepwise protonation of DHF by a water molecule prior to the hydride transfer. These two events are facilitated by two active site residues (D27 and Y100) that operate synergistically to ensure catalysis and to enable efficient DHF protonation over a wide pH range.