FIG 6 .

Validation of two-step HK autophosphorylation mechanism. Time course analysis of formation of labeled MtrB^H305Q·ATP complex using the HTA platform in the presence of various concentrations of unlabeled ATP as shown in the panels. (A) 5 µM, (B) 10 µM, (C) 100 µM, (D) 150 µM, (E) 200 µM. A fixed concentration of labeled ATP (20 nM) and 5 µM mutated MtrB^H305Q was used for all reactions. Best-fits (solid lines) along with 95% confidence intervals (dashed lines) of model predictions (equation 10) are obtained by simultaneous fitting to the data using k_p = 0 (absence of phosphorylation), K_e = 1.7 mm⁻¹, and ϕ as adjustable parameter. The data are shown as means ± SD (n = 3).