TABLE 2.

Determination of the kinetic parameters for the acylation step for R124L, R301L, and R124L/R301L MSDHs under pre-steady-state conditions with MMSA as substrate

Pre-steady-state measurements were performed at 30 °C in 50 mm potassium phosphate buffer (pH 8.2). Mutated MSDHs were preincubated with 2 mm NAD (R301L MSDH) or with 2 mm NAD and 500 μm CoA (R124L and R124L/R301L MSDHs) at 30 °C prior to making the kinetic measurements. The k₂ values of the acylation step were obtained by linear regression of k_obs determined under subsaturating concentrations of substrate MMSA (up to 16 mm). For the wild-type MSDH, the second-order rate constant k₂ was obtained by dividing k_{obs max} by K_app.

	Kapp MMSA	kobsmax MMSA	k2 MMSA
	mm	s−1	m−1 s−1
Wild typea	3.5 ± 1.0	1200 ± 180	3.4 × 105
R124L	>15		6 × 103
R301L	>15		5.4 × 103
R124L/R301L	>15		6.5 × 102

^a Data are from Ref. 25.