Table 1.

Steady-state parameters for amino acid activation by wild-type (WT) EcLeuRS^a

AA	AA structure	Km (AA)	kcat	kcat/Km	Discrimination factorb
		mM	s−1	mM−1s−1
WT LeuRS
Leuc		0.05 ± 0.01	66 ± 2	1,320
Nvac		4.9 ± 0.4	56 ± 1	11.4	116
Abad		85 ± 9	20 ± 1	0.24	5,500
Ilee,g		26 ± 7	1.1 ± 0.1	0.042	31,429
M40G LeuRS
Leuf		0.34 ± 0.03	6.9 ± 0.2	20
Nvaf		17 ± 3	4.2 ± 0.3	0.247	81
Ilee, g		10 ± 2	1.14 ± 0.06	0.114	175

The values represent the best fit value ± s.e.m. of at least two independent experiments.

^aMeasured by ATP-PP_i exchange assay.

^bDiscrimination factor is defined as (k_cat/K_m)_cognate/(k_cat/K_m)_non-cognate.

^cParameters reported in (Cvetesic et al, 2012).

WT or M40G LeuRS were used at ^d10 nM, ^e150 nM, or ^f50 nM concentration, and Leu, Nva, and Ile concentrations were varied over the range 0.1–10 times the K_m.

^gIsoleucine preparation used to determine activation parameters was purified as described in the Supplementary Materials and Methods.