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Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons98673

Ahmadian,  Mohammad Reza
Sonstige Wissenschaftliche Organisationseinheiten, Max Planck Institute of Molecular Physiology, Max Planck Society;

Hoffmann,  Ulrike
Max Planck Institute of Molecular Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons98693

Goody,  Roger S.
Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons98738

Wittinghofer,  Alfred
Sonstige Wissenschaftliche Organisationseinheiten, Max Planck Institute of Molecular Physiology, Max Planck Society;

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Citation

Ahmadian, M. R., Hoffmann, U., Goody, R. S., & Wittinghofer, A. (1997). Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy. Biochemistry, 36(15): 1, pp. 4535-4541. Retrieved from http://dx.doi.org/10.1021/bi962556y.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-1085-F
Abstract
Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Ras proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower phase, which corresponds to the hydrolysis reaction. With Ras bound to the nonhydrolyzable analogue mant-GppNHp, only the concentration-dependent first phase was observed. The Ras·mant-GppNHp·NF1-333 complexes were also used to measure dissociation rate constants of the Ras-GAP complexes. Using GAP-334 as the catalyst, the concentration-dependent first phase was too fast to be measured by the stopped-flow method, but the subsequent chemical cleavage reaction occurred at a similar rate (5-10 s-1) to that seen with NF1-333. With both GAP-334 and NF1-333, after rapidly reaching the initial equilibrium, there was no further time-dependent change on mixing GAPs with Ras·mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-catalyzed GTPase reaction on Ras. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.