Figure 6. Presteady State binding of PAP to SULT1A1(EGCG)₂.

A.) k_obs vs [PAP]. The binding- reaction progress curves were monitored using a stopped-flow fluorimeter (λ_ex = 290 nm, λ_em ≥ 325 nm (cutoff filter). k_obs values (black dots) were obtained by fitting 6–9 averaged progress curves using a single-exponential equation. Each kobs value is the average of three independent determinations. Red dots indicate k_obs values predicted using the k_on and k_off values obtained from the experiments associated with Panels B and C. B.) Binding at the High-Affinity Site (k_obs vs [PAP]). Reactions were initiated by mixing (1:1) a solution containing SULT1A1 (75 nM, dimer), EGCG (12 μM, 18 ▪ K_{d cap open form}), MgCh₂ (5.0 mM), NaPO₄ (50 mM), pH 7.5, 25 ± 2°C, with a solution that was identical except that it lacked SULT1A1 and contained PAP at the indicated concentrations. C.) Binding at the Low-Affinity Site (k_obs vs [PAP]). Reactions were initiated by rapidly mixing (1:1) a solution containing SULT1A1 (1.0 μM, dimer), 1.0 μM PAP (42 ▪ K_{d high affinity} and 1.3 ▪ K_{d low affmity}), EGCG (12 μm, 18 ▪ K_{d cap open form}), MgCl₂ (5.0 mM), NaPO₄ (50 mM), pH 7.5, 25 ± 2°C, with a solution that was identical except that it lacked SULT1A1 and contained PAP at the indicated concentrations. Pre-equilibration at [PAP] = [SULT1A1]_dimer saturates the high-affinity nucleotide-binding site and thus prevents it from contributing to the low-affinity binding measurements.