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Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH2 and GTPγF

MPS-Authors

Stumber,  Michael
Max Planck Institute of Molecular Physiology, Max Planck Society;

Herrmann,  Christian
Max Planck Institute of Molecular Physiology, Max Planck Society;

Wohlgemuth,  Sabine
Max Planck Institute of Molecular Physiology, Max Planck Society;

Kalbitzer,  Hans Robert
Max Planck Institute of Molecular Physiology, Max Planck Society;

Jahn,  Werner
Max Planck Institute of Molecular Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons98692

Geyer,  Matthias
Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society;

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Citation

Stumber, M., Herrmann, C., Wohlgemuth, S., Kalbitzer, H. R., Jahn, W., & Geyer, M. (2002). Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH2 and GTPγF. European Journal of Biochemistry, 269(13): 1, pp. 3270-3278. Retrieved from http://dx.doi.org/10.1046/j.1432-1033.2002.03003.x.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-0E53-1
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 DeltaHdouble dagger = 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 theGTP- 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 studie