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

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.

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Genre: Journal Article
Alternative Title : Biochemistry

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 Creators:
Ahmadian, Mohammad Reza1, Author           
Hoffmann, Ulrike2, Author
Goody, Roger S.3, Author           
Wittinghofer, Alfred1, Author           
Affiliations:
1Sonstige Wissenschaftliche Organisationseinheiten, Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753294              
2Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753286              
3Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753289              

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Free keywords: BINDING-SITE CATALYTIC DOMAIN ESCHERICHIA-COLI FLUORESCENCE GAP GTPASE-ACTIVATING PROTEIN HYDROLYSIS KINETICS NEUROFIBROMATOSIS TYPE-1 GENE P21 PRODUCT PROTEIN PROTEINS SPECTROSCOPY
 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.

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Language(s): eng - English
 Dates: 1997
 Publication Status: Issued
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 42878
URI: http://dx.doi.org/10.1021/bi962556y
 Degree: -

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Title: Biochemistry
  Alternative Title : Biochemistry
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 36 (15) Sequence Number: 1 Start / End Page: 4535 - 4541 Identifier: -