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Schlagwörter:
nucleotides; nucleotide analogues; NMRspectroscopy; GTP hydrolysis; Ras
Zusammenfassung:
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
