Figure 7.

The depletion of ATP by PcrA, monitored by Rho-MatB. The measurement was done at 10 μM ATP, using 1 μM Rho-MatB at different concentrations of dT₂₀. Thus, the presence of the biosensor has little effect on the concentration of free ATP and, as the K_m for ATP with PcrA is ∼3 μM, the rates at saturating dT₂₀ can be close to maximum. (A) Time courses at nanomolar dT₂₀ concentrations shown. Data were converted from fluorescence to [ATP] using a calibration curve (inset), with identical solution conditions at the highest concentration of dT₂₀, fitted to a hyperbola so that the whole concentration range was accessible. (B) The rates obtained by linear regression were fitted to the Michaelis–Menten model. The average K_m and k_cat values (n = 3) are 82.5 ± 13.2 nM and 10.5 ± 0.6 s^–1.