title: 13C and 15N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase.
abstract: 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).
authors.affiliation: Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: B S
authors.initials: BS
authors.lastname: Selinsky
authors.forename: M E
authors.initials: ME
authors.lastname: Perlman
authors.forename: R E
authors.initials: RE
authors.lastname: London
authors.forename: C J
authors.initials: CJ
authors.lastname: Unkefer
authors.forename: J
authors.initials: J
authors.lastname: Mitchell
authors.forename: R L
authors.initials: RL
authors.lastname: Blakley
mesh_terms: D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D002417:Cattle; D005492:Folic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase