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Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH 2 and GTPγF
Ist Teil von
European journal of biochemistry, 2002-07, Vol.269 (13), p.3270-3278
Erscheinungsjahr
2002
Link zum Volltext
Quelle
Wiley Online Library Journals Frontfile Complete
Beschreibungen/Notizen
Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPγS 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
‐(γ‐amidotriphosphate) (GTPγNH
2
) and a new synthesis of guanosine 5′‐
O
‐(γ‐fluorotriphosphate) (GTPγF). 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 GTPγNH
2
the enthalpy of magnesium coordination is Δ
H
° = 3.9 kcal·mol
−1
and the association constant
K
a
is 0.82 m
m
−1
. The activation energy for GTPγNH
2
·Mg
2+
complex formation is Δ
H
‡
= 7.8 ± 0.15 kcal·mol
−1
, similar to that for the natural substrate GTP. For GTPγF we obtained a similar enthalpy of Δ
H
° = 3.9 kcal·mol
−1
while the magnesium association constant is only
K
a
= 0.2 m
m
−1
. The application of both guanine nucleotide analogues to theGTP‐binding protein Ras was investigated. The rate of hydrolysis of GTPγNH
2
bound to Ras protein lay between the rates found for Ras‐bound GTPγS and GppNHp, while Ras‐catalysed hydrolysis of GTPγF 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.