TABLE 1.

Kinetic parameters of the reactions catalyzed by wild-type and mutated MSDHs under steady-state conditions with MMSA as substrate

Kinetic parameters were deduced from nonlinear least square regression of experimental data sets, according to the Michaelis-Menten equation. All K_m values were determined at saturating concentrations of the other substrates, and k_cat values are expressed per active subunit (i.e. two active subunits per tetramer). The steady-state initial rates of the reaction of mutated MSDHs were measured at 30 °C in 50 mm potassium phosphate buffer (pH 8.2), under similar conditions as those for the wild-type MSDH. Mutated MSDHs were preincubated with 2 mm NAD or with 2 mm NAD and 500 μm CoA at 30 °C, prior to making the kinetic measurements.

	Km MMSA	Km CoAa	Km NADa	kcatb
	mm	μm	mm	s−1
Wild typec	0.06 ± 0.01	120 ± 20	2.30 ± 0.06	2.2 ± 0.2
R124L	0.006 ± 0.001	570 ± 70	0.06 ± 0.01	0.060 ± 0.01
R301L	0.023 ± 0.005	620 ± 80	0.12 ± 0.01	0.14 ± 0.02
R124L/R301L				<10−2d

^a After dilution of the preincubated enzymes, the remaining NAD (12 μm) and CoA (3 μm) concentrations were taken into account for determination of the K_m values.

^b For all MSDHs, the rate-limiting step remains associated with the so-called “deacylation” step (i.e. all the steps occurring after hydride transfer).

^c Data are from Ref. 25.

^d Due to lack of saturation with substrates and very low steady-state initial rates (<10⁻² s⁻¹), it was not possible to determine the k_cat and K_m values.