Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.

ABSTRACT: Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex

SUBMITTER: Luo R 

PROVIDER: S-EPMC1863566 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature