13C and 15N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase.

The state of protonation of substrates bound to mammalian dihydrofolate reductase (DHFR) has significance for the mechanism of catalysis. To investigate this, dihydrofolate and dihydropteroyl-pentaglutamate have been synthesized with 15N enrichment at N-2. 15N NMR studies have been performed on the binary complexes formed by bovine DHFR with these compounds and with [5-15N]dihydrobiopterin. The results indicate that there is no protonation at N-5 in the binary complexes, and this was confirmed by 13C NMR studies with folate and dihydrofolate synthesized with 13C enrichment at C-6. The chemical shift displacements produced by complex formation are in the same direction as those which result from deprotonation of the N-3/C-4-O "amide" group and are consistent with at least partial loss of the proton from N-3. This would be possible if, as crystallographic data indicate, there is interaction of N-3 and the 2-amino group of the bound ligands with the carboxylate of the active site glutamate residue (Glu30).