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 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.

Figure 1. Kinetic analysis of ASAP1-catalysed GTP hydrolysis on Arf

(A) Steady-state analysis of the reaction catalysed by His₁₀–[325–724]ASAP1. In these experiments, Arf1-GTP was monitored by fluorescence [26]. Reactions contained 400 pM His₁₀–[325–724]ASAP1 and 0.6–16 μM myrArf1-GTP. Reactions were initiated by the addition of ASAP1. Initial slopes of the change in fluorescence were determined. Reaction rates were calculated from the initial slope data. The plot of Arf1 concentration versus reaction rate was fitted to the Michaelis–Menten equation and used to estimate V_max and K_m. (B) Single-turnover kinetic analysis of the reaction catalysed by His₁₀–[325–724]ASAP1. myrArf1-GTP was rapidly mixed with the indicated concentrations of [325–724]ASAP1. Reactions were quenched by rapid mixing with formic acid at the indicated times. The data were fitted to first- order rate equations. (C) Replot of single-turnover kinetic analysis to determine k_cat. The rates estimated from fitting the data in (B) were plotted against the concentration of ASAP1 added to the reaction. The maximum rate, k_cat, was estimated by fitting these data to the equation: Representative experiments of three are shown.

Figure 2. Mutants analysed in the present studies

Homology structure of Arf GAP and ANK domains of ASAP1 with mutated residues indicated.

Figure 3. Comparison of enzymatic power of mutants of His₁₀–[325–724]ASAP1

(A) Wild-type His₁₀–[325–724]ASAP1 was titrated into a reaction containing myrArf1-GTP. Reactions were stopped after 3 min by dilution with 20 mM Tris (pH 8.0), 10 mM MgCl₂, 100 mM NaCl and 1 mM DTT at 4 °C. Protein-bound nucleotide was trapped on nitrocellulose, eluted with formic acid and separated by TLC. (B) Effect of changes in Arg-497 of ASAP1. The experiment was performed as described in (A). Note the difference from (A) in both scales with up to 60 μM protein and less than 50% of the GTP hydrolysed. Results are from three experiments.

Figure 4. Steady-state kinetic analysis of His₁₀–[D512A,325–724]ASAP1

MyrArf1-GTP was titrated into a reaction initiated by the addition of 50 nM His₁₀–[D512A, 325–724]ASAP1. myrArf1-GTP was monitored by fluorescence. Initial rates were estimated from the time course data. A plot of initial velocity versus myrArf1-GTP concentration is shown. The results were fitted to the Michaelis–Menten equation to obtain estimates of K_m and V_max. The k_cat was calculated from the V_max and enzyme concentration. The experiment shown is representative of three.

Figure 5. Association of Arf1-GTP with ASAP1 with changes in Arg-497

(A) Inhibition of GAP activity by wild-type ASAP1. His₁₀–[R497A,325–724]ASAP1 or His₁₀–[R497Q,325–724]ASAP1 were titrated into a reaction containing 0.5 nM His₁₀–[325–724]ASAP1 and approx. 10 nM Arf1-GTP. Reaction rates are plotted against concentration of inactive ASAP1 mutant. Assuming the ASAP1 mutants inhibited by sequestering the substrate, the affinity for substrate is the K_i calculated from fitting the data to the equation k_obs=k_max/(1+mutant/K_i). (B) Arf1-GTP[S] binding to GST–[275–724]ASAP1. myrArf1–GTP[S] (μM) was incubated with 10 μM GST–[275–724]ASAP1 or the indicated mutants. Protein association was determined as described in the Experimental section. The results presented are the average for three experiments.

Figure 6. Analysis of single-turnover kinetics for His₁₀–[D512A,325–724]ASAP1

Reactants were rapidly mixed and the reaction quenched as described in the legend of Figure 1(B) and the Experimental section. This Figure shows a representative experiment of three performed using [D512A,325–724]ASAP1. (A) Time course for GTP hydrolysis with various ASAP1 concentrations. (B) Plot of k_obs determined from (A) against ASAP1 concentration.

Figure 7. Effect of premixing ASAP1 with lipid on single-turnover reaction rates

(A) Kinetic schemes. E represents the Arf GAP–ASAP1, S is Arf1-GTP and P is Arf1-GDP. Asterisk indicates a distinct conformational state. (B) [L8K]Arf1-GTP as substrate. Either His₁₀–[325–724]ASAP1 or [L8K]Arf1-GTP were premixed with vesicles, as indicated, prior to rapid mixing and quenching of the reaction. (C) myrArf1-GTP as substrate. myrArf1-GTP, prepared with LUVs (myrarf1-GTP is not stable without a hydrophobic surface), was rapidly mixed with His₁₀–[325–724]ASAP1 that had been premixed with LUVs. Reactions were quenched after the indicated incubation times. (D) Effect of Asp-512 on the lipid association step in the GAP reaction. This experiment was performed identically with the experiment described in (A) but used His₁₀–[D512A,325–724]ASAP1 as the GAP. The results shown are from three independent experiments.

Figure 8. Mutants of ASAP1 in cells

(A) Distribution of ASAP1. The indicated mutants of FLAG-tagged ASAP1 were expressed in NIH 3T3 fibroblasts. GFP was expressed in control cells. After 24 h, the cells were reseeded on fibronectin-coated coverslips, incubated for 5 h in serum-free medium and treated for 5 min with 60 ng/ml PDGF. Cells were fixed. Actin was visualized with rhodamine–phalloidin and ASAP1 visualized by staining for the FLAG epitope. (B) Effect on dorsal ruffle formation. Results are the percentage of cells expressing the indicated proteins with dorsal ruffles after a 9 min treatment with 20 ng/ml PDGF. Results are from four experiments, with 50 cells scored per condition in each experiment. (C) Co-localization with Arf1. ASAP1 was co-expressed with Arf1–HA in NIH 3T3 fibroblasts. Cells were treated as described in (A) but Arf1 and ASAP1 were visualized by immunostaining.