Equilibrium and kinetic study of the conformational transition toward the active state of p21Ha-ras, induced by the binding of BeF3- to the GDP-bound state, in the absence of GTPase-activating proteins.
Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-)ras to demonstrate that BeF3- binds to the GDP. p21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (DeltaH = 22.3 +/- 1.6 kJ mol-1). The similarity between the GTP-bound form and the GDP.BeF3--bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15 +/- 2 kJ mol-1 and DeltaH2 = 8 +/- 2 kJ mol-1). The kinetically determined enthalpy change of 23 +/- 4 kJ mol-1 is in excellent agreement with the equilibrium analysis.
Díaz
J F
JF
Laboratorium voor Chemische en Biologische Dynamica, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium.
Sillen
A
A
Engelborghs
Y
Y
D000838:Anions / Q000378:metabolism; D001608:Beryllium / Q000378:metabolism*; D017022:Flow Injection Analysis; D005459:Fluorides / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000838:Anions; D006150:Guanine Nucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; C035982:beryllium fluoride; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras); D001608:Beryllium; D005459:Fluorides