FIGURE 4.

Activation of apoACS. A, time dependent activation of apoACS in the presence of AcsF_Ch, MgATP, and nickel. 20 μm apoACS were incubated with 40 μm AcsF_Ch, 10 mm MgATP, and 40 μm NiCl₂ at 60 °C. Aliquots were taken from the activation assay at the indicated time points and the specific activity was measured at 25 °C by following the conversion of Mecob(III)inamid to cob(I)inamid by UV/visual spectroscopy. Assay conditions included a sample of the activation assay, Ti(III)citrate, Mecob(III)inamid, CO, and CoA in 0.1 m HEPES, pH 7.2. Each data point was measured in triplicate and error bars represent the standard deviation. Four control experiments were performed, in which AcsF_Ch, MgATP, NiCl₂, or apoACS was omitted from the activation assay. Data points for the control experiments were measured once. A monoexponential function was fitted to the data points giving an observed rate constant of 1.47 h⁻¹ for the activation. B, activation of apoACS after 60 (dark) and 180 min (light) in the presence of AcsF_Ch, K10A-AcsF_Ch, C108A-AcsF_Ch, CooC1_Ch, or CooC3_Ch, which was measured once. The inset shows a magnification of the data points for K10A-AcsF_Ch, C108A-AcsF_Ch, CooC1_Ch, and CooC3_Ch.