Proton, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-NADP-folate complex: characterization of three coexisting conformational states

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Abstract
The Lactobacillus casei dihydrofolate reductase-folate-NADP+ complex is shown by 1H and 13C NMR to exist in 3 interconverting conformational states, I, IIa and IIb. The proportions of the 3 states, as estimated from the intensities of the 3 separate 13C resonances observed in the complex containing [3-carboxamido-13C]NADP+, are pH dependent. State I predominates at low pH, and states IIa and IIb predominate at high pH; the ratio IIa:IIb is pH independent. The pH dependence of the interconversion of states I and IIa + IIb can be explained by a model in which a group on the enzyme has a pK of < 5 in state IIa + IIb and > 7 in state I. 1H, 13C and 31P NMR were used to characterize the structural differences between the 3 states of the complex. As judged by the 1H and 13C chemical shifts of the bound coenzyme, states I and IIa are similar to one another, but quite different from state IIb. This difference appears to be a localized one, since only the nicotinamide 2 and 4 protons, nicotinamide 3-carboxamide 13C and pteridine 7 proton show differences in chemical shift between these states. These differences are large, up to 1.4 ppm for 1H and 2 ppm for 13C. The remaining coenzyme protons and the 3 31P nuclei are unaffected. Studies of the C2 proton resonances of the 7 histidine residues show that the ionizable group responsible for the interconversion of states I and IIa + IIb is not a histidine (although 2 histidines show slight differences in environment between states IIa and IIb); the possible identity of this ionizable group and the nature of the conformational differences between the states are discussed.