TABLE 2.

Kinetic parameters of wild-type and mutant variants of MtNadD

Apparent kinetic parameters k_cat and K_m NaMN were determined in the presence of saturation ATP (1.5 mm) using the discontinuous coupled assay; the apparent K_m, ATP was determined at saturating NaMN (2–3 mm) using the end-point malachite green assay.

Mutations in MtNadD	kcat	Km NaMN	Km ATP	kcat/Km, NaMN	kcat/Km, ATP	IC50, 1594
	s−1	mm	mm	s−1mm−1	s−1mm−1	μm
None	5.5 ± 0.5	0.48 ± 0.06	0.041 ± 0.007	12	134	35 ± 11
T12A	0.22 ± 0.02	0.53 ± 0.13	0.042 ± 0.006	0.4	5	24 ± 2
D14A	<LODa
H17A	<LOD
H20A	<LOD
P44A	0.36 ± 0.02	0.84 ± 0.13	0.017 ± 0.003	0.4	21	37 ± 12
W45A	0.37 ± 0.03	0.98 ± 0.20	0.011 ± 0.003	0.4	34	56 ± 15
Q46Ab	4.7 ± 0.4	0.33 ± 0.09	0.033 ± 0.007	14	142	NDc
K47A	0.02 ± 0.002	2.70 ± 0.38	0.006 ± 0.001	0.007	3	94 ± 17
T86A	0.2 ± 0.02	2.80 ± 0.72	0.011 ± 0.2	0.07	18	114 ± 12
W117A	0.002 ± 0.0002	2.67 ± 0.50	-d	0.0008		30 ± 13
W117F	0.03 ± 0.008	1.13 ± 0.70	0.023 ± 0.006	0.03	1.30	ND
L164A	0.13 ± 0.01	0.052 ± 0.027	0.17 ± 0.03	2.5	0.8	ND
L164Q	0.015 ± 0.002	0.43 ± 0.13	0.19 ± 0.07	0.04	0.08	ND

^a LOD, limit of detection, is calculated as ∼0.0002 s⁻¹ under assay conditions.

^b Mutant enzyme preparation was only partially purified.

^c ND, not determined.

^d Value could not be reliably measured.