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          Institute: MPI für molekulare Physiologie     Collection: Sonstige wissenschaftliche Organisationseinheiten     Display Documents



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ID: 18884.0, MPI für molekulare Physiologie / Sonstige wissenschaftliche Organisationseinheiten
Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH2 and GTPγF
Authors:Stumber, Michael; Herrmann, Christian; Wohlgemuth, Sabine; Kalbitzer, Hans Robert; Jahn, Werner; Geyer, Matthias
Language:English
Research Context:nucleotide analogues
Date of Publication (YYYY-MM-DD):2002-07
Title of Journal:European Journal of Biochemistry
Journal Abbrev.:Eur. J. Biochem.
Volume:269
Issue / Number:13
Start Page:3270
End Page:3278
Sequence Number of Article:1
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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
Free Keywords:nucleotides; nucleotide analogues; NMRspectroscopy; GTP hydrolysis; Ras
External Publication Status:published
Document Type:Article
Communicated by:Jürgen Block
Affiliations:MPI für molekulare Physiologie/Abteilung I - Strukturelle Biologie/AG Struktur-Funktions-Beziehungen: PD Dr. Christian Herrmann
MPI für medizinische Forschung
MPI für molekulare Physiologie/Abteilung III - Physikalische Biochemie/AG Molekulare Mechanismen viraler Pathogenese: Dr. Matthias Geyer
Identifiers:URL:http://dx.doi.org/10.1046/j.1432-1033.2002.03003.x [article locator with full text links]
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