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

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Cell Signaling

Background:

• ADP-ribosylation factor GTPase-activating proteins (GAPs) regulate small GTP-binding proteins, including Arf proteins.
• ASAP1, an Arf GAP, has potential roles as an effector and oncogene, necessitating kinetic characterization.
• Understanding Arf GAP kinetics is vital for deciphering Arf-mediated cellular processes.

Purpose of the Study:

• To kinetically characterize the GTPase-activating protein ASAP1 using a truncated form.
• To investigate the mechanism of Arf1-GTP hydrolysis catalyzed by ASAP1.
• To explore the functional significance of specific ASAP1 residues in catalysis and Arf binding.

Main Methods:

• Steady-state and single-turnover kinetics were employed to determine kinetic parameters (kcat, Km) of ASAP1 with Arf1-GTP.
• Site-directed mutagenesis was used to probe the roles of specific ASAP1 residues (Arg-497, Trp-479, etc.).
• An in vivo assay measuring dorsal ruffle formation was utilized to assess functional consequences of mutations.

Main Results:

• ASAP1 exhibited Michaelis-Menten kinetics with Arf1-GTP, yielding kcat and Km values.
• Tetrafluoroaluminate stabilized a complex between Arf1-GDP and ASAP1, suggesting a transition state intermediate.
• Mutations primarily affected kcat, not Km, indicating roles in catalysis rather than substrate binding, and in vitro inactivity correlated with in vivo functional loss.

Conclusions:

• ASAP1 functions in a binary complex with Arf1-GTP to facilitate GTP hydrolysis, supporting a transition state model.
• Specific mutations in ASAP1 significantly impair its catalytic activity, impacting its in vivo function.
• The findings suggest a conformational change in the Arf1-GTP-ASAP1 complex during catalysis, with implications for ASAP1's effector role.