TABLE 3.

Kinetic parameters for the reactions catalyzed by the wild-type and mutated MSDHs under steady-state conditions with PPA 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 wild-type and mutated MSDHs were measured at 30 °C in 50 mm potassium phosphate buffer (pH 8.2). Wild-type and mutated MSDHs were preincubated with 2 mm NAD at 30 °C before kinetic measurements. ND, not determined.

	Km PPA	Km CoA	Km NADa	kcatb	kcat/Km CoA
	mm	μm	μm	s−1	m−1 s−1
Wild type	9.8 ± 0.3	85 ± 10	33 ± 13	12.6 ± 0.2	1.5 × 105
R124L	9.3 ± 1.1	330 ± 35	38 ± 1	2.8 ± 0.4	8.5 × 103
R301L	ND	>1000	ND	>3	3.0 × 103c

^a After dilution of the preincubated enzyme, the remaining NAD concentration (2 μm) was 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 This value is only an estimate, since the kinetic parameters cannot be determined with accuracy due to lack of saturation with CoA.