title: Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.
abstract: Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.
authors.affiliation: Max-Planck-Institute for Medical Research, Department of Biophysics, Heidelberg, Federal Republic of Germany.
authors.forename: H R
authors.initials: HR
authors.lastname: Kalbitzer
authors.forename: J
authors.initials: J
authors.lastname: Feuerstein
authors.forename: R S
authors.initials: RS
authors.lastname: Goody
authors.forename: A
authors.initials: A
authors.lastname: Wittinghofer
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D001411:Geobacillus stearothermophilus / Q000032:analysis*; D006153:Guanosine Diphosphate / Q000302:isolation & purification; D006160:Guanosine Triphosphate / Q000302:isolation & purification*; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification*; D010710:Phosphates / Q000302:isolation & purification
chemical_list: D010103:Oxygen Isotopes; D010710:Phosphates; D006153:Guanosine Diphosphate; D008345:Manganese; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D010100:Oxygen