FIGURE 7.

Steady-state kinetics and competitive inhibition of PqsBC by 2-AA. A, conversion of 2-ABA (λ_max at 360 nm) and octanoyl-CoA (absorbing at 260 nm) to HHQ (λ_max at 313 nm). 20 nm PqsBC was mixed with 50 μm of each substrate. Spectra were measured every 60 s with a bandwidth of 1 nm. HHQ formation could be observed at 313 nm with a differential extinction coefficient of 6,520 m⁻¹ cm⁻¹. The remaining absorption above 340 nm after full conversion of the substrates (bold trace) is due to scattering and can be eliminated by diluting to 50% in 2-propanol/HCl (dashed trace). Inset, conversion of 10 μm substrates, monitored at 313 nm (HHQ) and 360 nm (2-ABA) with the determined extinction coefficients. Data interval is 1 min. B–D, inhibition of PqsBC activity by 2-AA. B, nonlinear fits of measured initial rates. The Lineweaver-Burk plot (C) shows the characteristic pattern of competitive inhibition. Using the Dixon plot (D), the inhibitor constant K_i of the competitive reaction could be determined graphically. Experiments were performed with 50 μm octanoyl-CoA and 20 nm PqsBC in HEPES buffer, pH 8.2, at 25 °C; arrows indicate ascending concentrations.