FIGURE 4.

Rates of guanine nucleotide binding and hydrolysis by affinity-enhanced Gα_i1 mutants. A, nucleotide binding assays were initiated by the addition of [³⁵S]GTPγS to 100 nm WT or mutant Gα_i1 protein in either assay buffer or nonspecific buffer (the latter with excess unlabeled nucleotide). At the indicated time points, 100 μl of the protein mixture was vacuum-filtered through nitrocellulose, washed four times, and counted by scintillation. Assays were conducted in duplicate. The error bars represent S.E. Rates of binding (k_obs) were determined using nonlinear regression to be 0.07 min⁻¹ (95% CI, 0.04–0.10), 0.04 min⁻¹ (95% CI, 0.02–0.05), 0.06 min⁻¹ (95% CI, 0.04–0.09), and 0.07 min⁻¹ (95% CI, 0.06–0.08) for Gα_i1^WT, Gα_i1^E245L, Gα_i1^E116L, and Gα_i1^Q147L, respectively. B, intrinsic GTPase activities of WT and mutant Gα_i1 subunits were determined using [γ-³²P]GTP single-turnover assays. 100 nm Gα_i1 protein was loaded with [γ-³²P]GTP at 20 °C for 10 min before assays were initiated by the addition of Mg²⁺ (10 mm) and GTPγS (400 μm). At the indicated time points, 100 μl of the protein mixture was quenched with activated charcoal, and released [³²P]P_i was counted by scintillation. Assays were conducted in duplicate. The error bars represent S.E. GTPase rates (k_cat) were determined using nonlinear regression to be 0.48 min⁻¹ (95% CI, 0.40–0.56), 0.15 min⁻¹ (95% CI, 0.12–0.17), 0.58 min⁻¹ (95% CI, 0.46–0.70), and 0.65 min⁻¹ (95% CI, 0.50–0.81) for Gα_i1^WT, Gα_i1^E245L, Gα_i1^E116L, and Gα_i1^Q147L, respectively.