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

Abstract

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) (GTPγNH2) 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 31P, 19F and 1H NMR resonances are tabulated. For GTPγNH2 the enthalpy of magnesium coordination is ΔH° = 3.9 kcal·mol−1 and the association constant Ka is 0.82 mm−1. The activation energy for GTPγNH2·Mg2+ complex formation is ΔH‡ = 7.8 ± 0.15 kcal·mol−1, similar to that for the natural substrate GTP. For GTPγF we obtained a similar enthalpy of ΔH° = 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 theGTP‐binding protein Ras was investigated. The rate of hydrolysis of GTPγNH2 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.

ADP-ribosylation Factor 1-independent Protein Sorting and Export from the trans-Golgi Network

“…Nucleotide Dependence of HA Release-A number of GTPases have been implicated in the delivery of HA from the TGN to the plasma membrane, including ARF1 and G s ␣ (42,43). To determine whether there is a requirement for GTP or GTP hydrolysis in HA release, we tested the effect of poorly hydrolyzable GTP analogs in our assay (44). Inclusion of GTP␥S in the reconstitution reaction decreased HA release in a dose-dependent manner with complete inhibition of ATP-dependent release observed at 100 M concentration (Fig.…”

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

“…Adenosine 5′‐ O ‐fluorophosphate (F‐pA) was synthesized according to [23] and adenosine 5′‐ O ‐(γ‐fluorophosphate) (Fpp‐pA) according to procedure developed for the synthesis of GTPγF [24]. Other adenine (di)nucleotides were from Sigma, St. Louis, MO, USA.…”

A survey of the year 2002 literature on applications of isothermal titration calorimetry

“…We have sorted these arbi-trarily according to the binding partner for the protein, i.e. small molecules (physiological ligands), [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] drug/inhibitor (natural and synthetic inhibitors), 37-44 other proteins (or peptides derived thereof), nucleotides, 72-77 metal ions, 78-89 polysaccharides [90][91][92][93][94][95][96] and lipids. [97][98][99] Finally some additional miscellaneous studies reported on some surface chemistry 100,101 and protein-folding studies were published.…”