FIGURE 6.
pH dependence of the acylation rate constant kac for the R124L MSDH-catalyzed reaction with PPA as substrate. Pre-steady-state data were collected at 30 °C on an SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) using the 20-μl cell, by rapidly mixing 16 μn R124L MSDH (expressed in subunits), 1 mm NAD, 0.5 m PPA, and 500 μm CoA (final concentrations) over a pH-range of 5.50–8.75 in buffer A. Following excitation at 295 nm, formation of NADH was measured by monitoring the fluorescence emission using a 395-nm cutoff filter. Each individual point is the average of at least six measurements with an error of <5%. Experimental data (●) were fit by nonlinear least square regression analysis against a two-pKa model, which gave pKapp values of 5.7 ± 0.1 and 7.5 ± 0.2 and rate constants kac (i) of 67 ± 4 and kac (max) of 92 ± 2 s−1, respectively (solid line). The data do not fit to a one-pKa model (dotted line).