FIGURE 3.

Determination of the catalytic parameters for the acylation step of wild-type MSDH in the presence of PPA, under pre-steady-state conditions. The appearance of NADH was selectively monitored using FRET on an SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) at 30 °C. One syringe is filled with 16 μn wild-type MSDH (expressed in subunit), 1 mm NAD, and 500 μm CoA (final concentrations), and the other one contained PPA at various concentrations. Following selective excitation of Trp residues at 295 nm, the fluorescence emission of NADH was measured using a 395-nm cutoff filter. Rate constants k_obs were fit to Equation 1, which gave a k_{obs (max)} of 340 ± 20 s⁻¹ and a K_app for PPA of 0.15 ± 0.02 m. The inset shows the results obtained in the absence of CoA or desulfo-CoA.