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Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPcS 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-(c-amidotriphosphate) (GTPcNH2) and a new synthesis of guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on 31P, 19F and 1H NMR resonances are tabulated. For GTPcNH2 the enthalpy of magnesium coordination is DH° ¼ 3.9 kcalÆmol)1 and the association constant Ka is 0.82 mM)1 | Eur. J. Biochem. 269 3270-3278 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03003.x Synthesis characterization and application of two nucleoside triphosphate analogues GTPỵNH2 and GTPyF Michael Stumber1 Christian Herrmann2 Sabine Wohlgemuth2 Hans Robert Kalbitzer1 Werner Jahn1 and Matthias Geyer 1 Max-Planck-Institut fur medizinische Forschung Department of Biophysics 69120 Heidelberg Germany 2Max-Planck-Institut fur molekulare Physiologie Department of Structural Biology 44227 Dortmund Germany Guanosine triphosphate nucleotide analogues such as GppNHp also named GMPPNP or GTPyS 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- y-amidotriphosphate GTPyNH2 and a new synthesis of guanosine 5 -O- y-fluorotriphosphate GTPyF . The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on 31P 19F and 1H NMR resonances are tabulated. For GTPyNH2 the enthalpy of magnesium coordination is AH 3.9 kcal-mol-1 and the association constant Ka is 0.82 mM-1. The activation energy for GTPyNH2-Mg2 complex formation is AH 7.8 0.15 kcal-mol-1 similar to that for the natural substrate GTP. For GTPyF we obtained a similar enthalpy of AH 3.9 kcal-mol-1 while the magnesium association constant is only Ka 0.2 mM-1.The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated. The rate of hydrolysis of GTPyNH2 bound to Ras protein lay between the rates found for Ras-bound GTPyS and GppNHp while Ras-catalysed hydrolysis of GTPyF 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. Keywords nucleotides nucleotide analogues NMR spectroscopy GTP hydrolysis Ras. Nucleotides are fundamental components in cellular metabolism. Acting as substrates for nucleotide .
