Figure 6

Mutational effects of GTSLPE proline and glycine on ion-exchange activities.

(A) The K_m values of the Na⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange reactions were measured by varying the concentrations of ⁴⁵Ca²⁺ (20–2000 μM) in the assay medium and using saturating concentrations of intravesicular Na⁺ (160 mM) or Ca²⁺ (250 μM), as described in Methods (see also Fig. S1). The bars represent the mean ± SE. (B) The k_cat values were calculated as k_cat = V_max/[E]_t, where [E]_t was normalized by using the GFP assay. The bars represent the mean ± SE. (C) The k_cat/K_m values were derived from experimentally observed k_cat and K_m values as outlined in panels (A) and (B). The bars represent the mean ± SE. (D) Experimentally derived values of k_cat and K_m of the Ca²⁺/Ca²⁺ exchange reaction were used for calculating the ∆∆G^≠_app values according to the equation ∆∆G^≠_app = −2.303RT•log[(k_cat/K_m)_mut/(k_cat/K_m)_wt. The bars represent the mean ± SE. (E) The difference between the HDX profile of the apo and Na⁺-bound forms of NCX_Mj at 1200 sec are overlaid on the crystal structure of NCX_Mj (PDB 3V5U). The color key indicates the difference in the percentage of deuterium incorporation between the Na⁺-bound and apo forms. The blue labeling corresponds to regions with less deuterium incorporation in the Na⁺-bound form compared with the apo form. The results are nearly identical for the Ca²⁺- and Na⁺-bound species.