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Title: Equilibrium and kinetic study of the conformational transition toward the active state of p21(Ha-ras), induced by the binding of BeF3- to the GDP-bound state in the absence of GTPase-activating proteins
Authors: Diaz, JF ×
Sillen, A
Engelborghs, Yves #
Issue Date: Jan-1997
Publisher: Amer soc biochemistry molecular biology inc
Series Title: Journal of Biological Chemistry vol:272 issue:37 pages:23138-23143
Abstract: Hitherto ras-related GTP-binding proteins have been considered not to hind 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 tee those of the active state, The binding has a low affinity (K-d at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (Delta H = 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 (Delta H-1 = 15 +/- 2 kJ mol(-1) and Delta H-2 = 8 +/- 2 kJ mol(-1)). The kinetically determined enthalpy change of 23 +/- 4 kJ mol(-1) is in excellent agreement with the equilibrium analysis.
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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