Figure 5. Effect of coatomer on kinetics of Arf GAP2-catalyzed GTP hydrolysis. Panels A – C. Saturation kinetics performed with fluorescent based assay.

The conversion of Arf1•GTP to Arf•GDP was followed using tryptophan fluorescence in a reaction containing LUVs with PI4P and extruded through 0.03 μm pores and either (A) 750 nM [1–521] Arf GAP2 His₆, (B) 5 nM coatomer with 200 nM [1–521] Arf GAP2 His₆ or (C) 124 nM coatomer with 114 nM [1–521] Arf GAP2 His₆. Initial rates were estimated, and the plot of initial rate versus myrArf1•GTP was fit to the Michaelis-Menten equation to estimate the K_m and V_max. The K_m and the k_cat, calculated from the V_max, are presented in Table 3. Panels D – F. Saturation kinetics performed following [α³²P]GTP hydrolysis. [α³²P]GTP•Arf1 was titrated into reactions containing LUVs with PI4P and extruded through 0.03 μm pores and either (D) 3.4 μM [1–521] Arf GAP2 His₆, (E) 5 nM coatomer with 100 nM [1–521] Arf GAP2 His₆ or (F) 124 nM coatomer with 10 nM [1–521] Arf GAP2 His₆ as indicated. Initial rates were estimated, and the plot of initial rate versus myrArf1•GTP was fit to the Michaelis-Menten equation to estimate the K_m and V_max. The K_m and k_cat, calculated from the V_max, are presented in Table 3. Panel G. Arf GAP2 concentration dependence in the presence of limiting coatomer. [1–521] Arf GAP2 His₆ was titrated into a reaction containing LUVs containing PI4P and extruded through 0.03 μm pores, 0.6 μM of [α³²P]GTP•myrArf1 and 20 nM coatomer. Reactions were stopped after 3 min at 30°C. Three experiments are summarized.