Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.
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 of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.
Luo
Ruibai
R
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Ahvazi
Bijan
B
Amariei
Diana
D
Shroder
Deborah
D
Burrola
Beatriz
B
Losert
Wolfgang
W
Randazzo
Paul A
PA
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D050356:Lipid Metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
D048868:Adaptor Proteins, Signal Transducing; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1