de.mpg.escidoc.pubman.appbase.FacesBean
English
 
Help Guide Disclaimer Contact us Login
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

Rap-specific GTPase activating protein follows an alternative mechanism

MPS-Authors

Brinkmann,  Thilo
Max Planck Institute of Molecular Physiology, Max Planck Society;

Daumke,  Oliver
Max Planck Institute of Molecular Physiology, Max Planck Society;

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

Kühlmann,  Dorothee
Max Planck Institute of Molecular Physiology, Max Planck Society;

Stege,  Patricia
Max Planck Institute of Molecular Physiology, Max Planck Society;

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

Ahmadian,  Mohammad Reza
Sonstige Wissenschaftliche Organisationseinheiten, 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;

Locator
There are no locators available
Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

Brinkmann, T., Daumke, O., Herbrand, U., Kühlmann, D., Stege, P., Ahmadian, M. R., et al. (2002). Rap-specific GTPase activating protein follows an alternative mechanism. Journal of Biological Chemistry, 277(15): 1, pp. 12525-12531. Retrieved from http://www.jbc.org/cgi/content/abstract/277/15/12525.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-0E95-D
Abstract
Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down- regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degreesC. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V-max of the GTP- hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP- catalyzed GTP-hydrolysis reaction on Rap1.