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Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states



Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states



Biochemistry 21(23): 5831-5838



The Lactobacillus casei dihydrofolate reductase-folate-NADP+ complex is shown by 1H and 13C NMR to exist in three interconverting conformational states, I, IIa, and IIb. The proportions of the three states, as estimated from the intensities of the three 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 less than 5 in state IIa + IIb and greater than 7 in state I. 1H, 13C, and 31P NMR has been used to characterize the structural differences between the three 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, however, large--up to 1.4 ppm for 1H and 2 ppm for 13C. The remaining coenzyme protons, as well as the three 31P nuclei, are unaffected. Studies of the C2 proton resonances of the seven histidine residues show that the ionizable group responsible for the interconversion of states I and IIa + IIb is not a histidine (although two 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.

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Accession: 043296016

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PMID: 6817782



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