Table S2.

Steady-state kinetic parameters for the RFK activity (RF + ATP → FMN + ADP) of the different CaFADS forms. Data obtained at 37 °C in 20 mM PIPES, 0.8 mM MgCl₂, pH 7.0.

	kcatb,c (min−1)	KmRF b,c (μM)	Kib,c (μM)	kcat/KmRF b (min−1 μM−1)	kcatc,d (min−1)	KmATP c,d (μM)	kcat/KmATP d (min−1 μM−1)
WT a	301	13	4.0	23	68	14	4.9
H28A	427	23	1.8	18	45	14	3.1
H28D	287	25	1.9	12	55	12	4.7
H31A	169	4.5	6.3	37	58	14	4.3
N125A	111	1.7	4.6	65	46	24	1.9
N125D	415	16	3.3	26	80	35	2.3
R161A	300	12	5.1	25	65	12	5.6
R161D	300	13	5.1	23	68	11	6.1
S164A	259	8.8	4.4	29	59	12	5.0
S164D	185	5.6	5.8	33	55	10	5.3
T165A	171	10	3.2	17	66	12	5.4
T165D	180	7.7	4.1	23	45	11	4.1

^aData from [24].

^bParameters determined at saturated ATP concentrations.

^cInhibition by substrate prevented the determination of true parameters and the values here reported correspond to apparent constants; ^appk_cat and ^appK_m. Estimated errors in ^appk_cat and ^appK_m values increased up to ±35%.

^dParameters estimated using a RF concentration exhibiting ~80% maximal activity before the maximum experimentally detected. Errors in k_cat and K_m were considered within ±10%, being this value taken larger than the standard deviation between three independent experiments and the numerical error after fitting analysis of each experiment to the Michaelis-Menten equation.