13C NMR determination of the tautomeric and ionization states of folate in its complexes with Lactobacillus casei dihydrofolate reductase

13C NMR studies provide a convenient way of obtaining detailed information about tautomeric and ionization states in protein-ligand complexes provided that suitably 13C-labeled molecules are available. In the present study, [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid were synthesized and the 13C NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analyzed as a function of pH. From these data it was possible to determine the tautomeric and ionization states of the bound folate and to obtain further evidence about the orientation of the pteridine ring in the complexes. In the 13C spectra of the ternary complexes of the 13C-labeled folic acids with DHFR and NADP+, each labeled carbon gave rise to multiple signals, confirming our previous findings that there are three interconverting conformational forms of bound folate (forms I, IIa, and IIb) in the ternary complex (Birdsall et al., 1989b). The 13C spectra of the binary complexes of folate and DHFR also provide direct evidence for the presence of forms IIa and IIb and indirect evidence of some form I at low pH values (<5.0). 2D 1H-13C HMQC- NOESY experiments on ternary complexes formed using the [2,4a,7,9-13C]folic acid were used to obtain intermolecular NOES between the folate H7 proton and protons on the protein, and these provided further characterization of the orientations of the pteridine ring in the different bound forms of folate (form IIb with its pteridine ring in the catalytically active conformation and forms I and IIa with their pteridine rings turned over by 180 degrees). The tautomeric and ionizable states of the pteridine rings in forms I, IIa, and IIb were investigated by comparing the 13C chemical shifts of the pteridine ring carbons in the different forms with values obtained from "model" compounds with known tautomeric and ionization states and by considering 1H chemical shifts of the enzyme complexes. Folate in form IIb, the catalytic active conformation of folate, exists in the keto form with N1 unprotonated whereas folate in form IIa and form I exists in enolic forms.