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. 2011 Apr 22;286(25):21971–21981. doi: 10.1074/jbc.M110.213280

FIGURE 2.

FIGURE 2.

Determination of the catalytic parameters of the acylation step for R124L, R301L, and R124L/R301L MSDH-catalyzed reactions under pre-steady-state conditions with MMSA as substrate. The appearance of NADH was monitored at 340 nm on an SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) at 30 °C. The final concentration of MSDH was 16 μn (expressed in subunit) with the MMSA concentration ranging from 1 to 16 mm. For each substrate concentration, experimental data were analyzed by nonlinear regression using the SX18MV-R software package to obtain kobs. As saturation with MMSA was not observed irrespective of the mutated MSDHs, k2 were extracted from the resulting linear equations. Key: R124L MSDH (●, solid line); R301L MSDH (▴, dashed line), R124L/R301L MSDH (■, solid line), and wild-type MSDH (♦, solid line, from Ref. 25). The inset details the results obtained on the three mutated MSDHs.