title: Variations in fluorescence and enzymic properties of bovine dihydrofolate reductase.NADPH complex during the slow conformational change induced by coenzyme binding.
abstract: When NADPH was added in large excess to bovine dihydrofolate reductase (H2folate reductase), there was a slow isomerization process between two conformers of the binary complex (B1<-->B2), as shown by changes in the fluorescence properties. Thus, we monitored the time dependence of (a) the quenching of protein intrinsic fluorescence intensity, (b) the polarization state of the fluorescence light emitted by NADPH.H2folate reductase complexes and (c), from a more biological point of view, the enzymic activity of binary complex solutions. The kinetics for these three processes were in good agreement using the same temperature conditions. Furthermore, fluorescence studies provided information on the NADPH environment in the binary complex. As soon as NADPH bound to H2folate reductase, light emitted by the invariant Trp24 residue located within the coenzyme-binding site was quenched by an energy-transfer process. Moreover, Trp57 and/or Trp113 emissions were partially quenched. The subsequent NADPH-bound protein conformational change caused an additional quenching, probably of Trp57 and/or Trp113 emissions. Thus, NADPH.H2folate reductase conformation was modified but no change was observed at the coenzyme-binding site, at least in our fluorescence study. These results were confirmed by polarization measurements. The conformational change, as well as the instantaneous NADPH binding, resulted in a more rigid form of the protein, as shown by an increase in steady-state anisotropy values. Finally, the isomerization process led to a more active enzymic form.
authors.affiliation: Equipe Protéines Membranaires, Groupe de Recherche sur les Interactions des Protéines en Pharmacologie, Faculté de Pharmacie, Marseille, France.
authors.forename: O
authors.initials: O
authors.lastname: Rimet
authors.forename: M
authors.initials: M
authors.lastname: Chauvet
authors.forename: M
authors.initials: M
authors.lastname: Dell'Amico
authors.forename: G
authors.initials: G
authors.lastname: Noat
authors.forename: M
authors.initials: M
authors.lastname: Bourdeaux
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005453:Fluorescence; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase