Determination by Raman Spectroscopy of the pK_a of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H₂folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pK_a of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pK_a of N5 of dihydrofolate in the Escherichia coli DHFR/NADP⁺/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pK_a of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP⁺. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H₂folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.