Fig. 7. The slow component in the tryptophan fluorescence quenching at basic pH. (A) Fluorescence quenching of 2 mM single Trp-63 at pH 8.5 after mixing with increasing concentrations of TPP⁺. The data are fitted to a second-order exponential equation. (B) The rate of the fast (filled symbols) and slow components (empty symbols) of the single Trp-63 mutant at pH 7.5 (squares), pH 8.0 (circles), and pH 8.5 (triangles). The magnitude of the slow component increases with pH but the rate of »1.5 sec^-1 is independent of pH and substrate concentration.

Fig. 8. Steady-state measurements of substrate-induced proton release using phenol red. (A) To increase the sensitivity of the measurements, phenol red was used as the only buffer of the solution, so that even small amounts of protons released from the protein promoted significant absorption changes. Three micromolar EmrE in a solution containing 0.15 M NaCl/0.08% DDM/100 mM phenol red were mixed with 5 mM TPP⁺. Decrease in the absorption at 556 nM represents acidification of the solution. Addition of NaOH reverses these absorption changes. These results are similar to the measurements done with a micro-pH electrode (1). The absorption ratio between the two peaks of phenol red absorption in 438 and 556 nm was used to determine the exact pH of each measurement. This method allowed us to measure the presteady-state protons release in the pH range from 6.5 to 7.7. (B) pH calibration curve of 100 mM phenol red solutions using the absorption ratio at 556/438 nm at the indicated pH values.

1. Soskine M, Adam Y, Schuldiner S (2004) J Biol Chem 279:9951-9955.

Fig. 9. Different substrates bind to EmrE and induce proton release at similar rates. Parallel measurements of the substrate binding and proton release of 1.5 mM EmrE were performed using TPP⁺ (A), TPMP⁺ (B) or ethidium⁺ (C) (similar to those presented in Fig. 4). All measurements were performed at pH »7.3. The K_on value presented in each figure is the one obtained for substrate binding. The K_i value is from ref. 2.

2. Rotem D, Steiner-Mordoch S, Schuldiner S (2006) J Biol Chem 281:18715-18722.

Fig. 10. Possible models that fits the pH dependence of the substrate-binding rates. (A) A model using equations adapted from ref. 3 assuming two different pK_as and three different rates for fully protonated protein [k_on(EH2)], protein that released one proton [kon(EH)], and deprotonated protein [kon(E)]. (B) Similar to A but assuming that the pK_as of the two groups are identical. (C) Similar to B but under the assumption that binding to protonated protein is impossible, therefore k_on (EH2) and k_on (EH) are both zero. (D) A model for protonation of a single group, under the assumption that the two protons leave the protein practically at the same time due to high cooperativity.

3. Fersht A (1985) Enzyme Structure and Mechanism (Freeman, New York).