title: Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPgammaNH(2) and GTPgammaF.
abstract: Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPgammaS are widely used to stabilize rapidly hydrolyzing protein-nucleotide complexes and to investigate biochemical reaction pathways. Here we describe the chemical synthesis of guanosine 5'-O-(gamma-amidotriphosphate) (GTPgammaNH(2)) and a new synthesis of guanosine 5'-O-(gamma-fluorotriphosphate) (GTPgammaF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on (31)P, (19)F and (1)H NMR resonances are tabulated. For GTPgammaNH(2) the enthalpy of magnesium coordination is DeltaH degrees = 3.9 kcal.mol(-1) and the association constant K(a) is 0.82 mm(-1). The activation energy for GTPgammaNH(2).Mg2+ complex formation is DeltaH++ = 7.8 +/- 0.15 kcal.mol(-1), similar to that for the natural substrate GTP. For GTPgammaF we obtained a similar enthalpy of DeltaH degrees = 3.9 kcal.mol(-1) while the magnesium association constant is only K(a) = 0.2 mm(-1). The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated. The rate of hydrolysis of GTPgammaNH(2) bound to Ras protein lay between the rates found for Ras-bound GTPgammaS and GppNHp, while Ras-catalysed hydrolysis of GTPgammaF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.
authors.affiliation: Max-Planck-Institut für medizinische Forschung, Department of Biophysics, Jahnstrasse 29, D-69120 Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Stumber
authors.forename: Christian
authors.initials: C
authors.lastname: Herrmann
authors.forename: Sabine
authors.initials: S
authors.lastname: Wohlgemuth
authors.forename: Hans Robert
authors.initials: HR
authors.lastname: Kalbitzer
authors.forename: Werner
authors.initials: W
authors.lastname: Jahn
authors.forename: Matthias
authors.initials: M
authors.lastname: Geyer
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002151:Calorimetry / Q000379:methods; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008670:Metals; C464798:guanosine 5'-O-(gamma-amidotriphosphate); C054439:guanosine 5'-(gamma-fluorotriphosphate); D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D008274:Magnesium