Figure 2.

Pre-steady-state binding of PAPS to SULT1A1. (A) Composite k_obs vs [PAPS] plot. Two well-isolated binding phases are observed. Binding was monitored via changes in SULT1A1 intrinsic fluorescence (λ_ex = 290 nm; λ_em ≥ 330 nm). k_obs values are the average of three independent determinations. Reaction conditions included SULT1A1 (0.050 μM, dimer), MgCl₂ (5.0 mM), NaPO₄ (50 mM), pH 7.2, and 25 ± 2 °C. Red dots indicate the k_obs values predicted using the k_on and k_off values obtained from the experiments associated with panels B and C. (B) k_obs vs [PAPS] for the high-affinity subunit. Reaction conditions were identical to those described for panel A except that [SULT1A1] = 0.030 μM (dimer). k_on = 2.0 ± 0.2 μM^–1 s^–1; k_off = 0.70 ± 0.02 s^–1. (C) k_obs vs [PAPS] for the low-affinity subunit. Reaction conditions were identical to those described for panel A except the SULT1A1 (2.0 μM, dimer) was equilibrated with PAPS [8.0 μM, 26K_{d(high affinity)}, 0.27K_{d(low affinity)}] before being mixed with PAPS at higher concentrations (20–80 μM). k_on = 0.96 ± 0.01 μM s^–1; k_off = 29 ± 1 s^–1. All reactions were pseudo-first-order in PAPS concentration.