Fig. 9.

Kinetic analysis of canonical and noncanonical substrate uptake by ABC transporters. (A) Kinetic scheme modeling canonical and noncanonical pathways in substrate import by the transporter, E, where E₁, E₂, E_2′, and E₃ represent different states of the transporter as shown in Fig. 8. The methionine ligand (m) binds to Q to form the liganded species Q_m with a dissociation constant K_d. Q_m and Q can bind to E₁ to form E_2′Q_m and E₂Q, with effective dissociation constants K₁ and K₂, respectively. Transport occurs from the state E₃Q•m that is generated either by isomerization of E_2′Q_m with the first-order rate constant k₁ or by binding of m to E₂Q with a second-order rate constant k₂. The steady-state solution to this scheme for the case where E, E_2′Q_m, E₂Q, Q, and Q_m are at equilibrium is described in Materials and Methods. (B) Dependence of the transport rate for a substrate at 10 µM concentration on various parameters of the kinetic scheme in A, as a function of the K_d for binding of substrate to the SBP. Curves i, ii, and iii correspond to k₂ = 5,000, 500, and 0 M⁻¹⋅s⁻¹, with Q_tot = 10⁻⁴ M, K₁ = K₂ = 10⁻⁴ M, and k = k₁ = 0.01 s⁻¹. For substrates with high affinity to the SBP (K_d < ∼10⁻⁶ M) the models are equivalent, whereas for more weakly bound substrates (K_d > ∼10⁻⁴ M) the transport rate depends critically on the value of k₂. Depending on the value of k₂, the rate of transport of a weakly bound substrate can exceed that of a substrate that binds tightly to the SBP (compare curves i and ii). When k₂ = 0 (curve iii), transport does not occur under these conditions, which corresponds to the case where substrate is only delivered to E₁ by binding to the SBP. (C) Dependence of the K_m for transport on the dissociation constant K_d for binding of substrate to the SBP. The red and black circles represent experimental data points for the methionine (this study) and maltose transporter (30), respectively. The curves are generated with the following parameters: i, k = 0.01 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 1,000 M⁻¹⋅s⁻¹; ii, k = 0.1 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 100 M⁻¹⋅s⁻¹; and iii, k = 0.1 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 0 M⁻¹⋅s⁻¹, with Q_tot = 10⁻⁴ M and K₁ = K₂ = 10⁻⁴ M for all curves. Curves were not explicitly fit to the experimental data but reflect parameter values that approximate the data. For this kinetic scheme, K_m reaches a plateau as K_d increases, and depending on the parameter values, this plateau can be tuned to the physiological concentration range. For curve iii, where substrate does not bind to E₂Q, a plateau region is not reached, so substrates with poor affinities for Q (high K_d) would have correspondingly high K_m values.