title: NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.
abstract: 5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR. The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase. The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed. The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR. The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour. Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way. Formation of the holoenzyme R1R2 also affects the equilibrium.
authors.affiliation: Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Université Joseph Fourier, Grenoble, France.
authors.forename: B
authors.initials: B
authors.lastname: Roy
authors.forename: J L
authors.initials: JL
authors.lastname: Decout
authors.forename: C
authors.initials: C
authors.lastname: Béguin
authors.forename: M
authors.initials: M
authors.lastname: Fontecave
authors.forename: P
authors.initials: P
authors.lastname: Allard
authors.forename: S
authors.initials: S
authors.lastname: Kuprin
authors.forename: A
authors.initials: A
authors.lastname: Ehrenberg
mesh_terms: D001665:Binding Sites; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005467:Floxuridine / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D012262:Ribonucleoside Diphosphate Reductase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: C092815:5-fluoro-2'-deoxyuridine-5'-diphosphate; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D005467:Floxuridine; C045813:2'-deoxycytidine diphosphate; D012262:Ribonucleoside Diphosphate Reductase