. Author manuscript; available in PMC: 2012 Mar 8.

Published in final edited form as: Biochemistry. 2011 Jan 31;50(9):1474–1482. doi: 10.1021/bi1013535

Table 2.

Steady State Kinetic Parameters for deacylation of AA-tRNA^Ala, at 37 °C and pH 7.5^a

AlaRS	seryl- tRNA^Ala; k_cat (s⁻¹)	seryl- tRNA^Ala; K_M (10⁻⁶ M)	seryl- tRNA^Ala; k_cat/K_M (10⁵ M⁻¹s⁻¹)	alanyl- tRNA^Ala; k_cat/K_M (10⁵ M⁻¹s⁻¹)	(k_cat/K_M)_Ser/ (k_cat/K_M)_Ala
WT	3.3±0.3	5.0±1.4	6.6	0.54	12.2
E664A	4.6±0.4	6.3±1.1	7.3	0.65	11.2
T567G	0.8±0.1	1.9±0.3	4.5	0.49	9.2
Q584N	N.D.^b	N.D.	6.1	5.0	1.2
Q584A	N.D.	N.D.	2.9	1.4	2.1
Q584H	N.D.	N.D.	1.1	0.57	1.9
I677E	N.D.	N.D.	1.0	0.67	1.5
C666A	N.D.	N.D.	0.44	0.74	0.6

For the WT, T567, and E664 enzymes with the non-cognate seryl-tRNA^Ala editing substrate, the k_cat and K_M values were obtained from the Michaelis-Menten plot. For the I677E, C666A, Q584H, Q584N, and Q584A AlaRS mutants, the k_cat/K_M constants were derived from the slopes of the V versus [tRNA] plots. For all experiments employing the cognate alanyl-tRNA^Ala as substrate, the k_cat/K_M constants were derived from the slopes of the V versus [tRNA] plots.

N.D., not determined, owing to failure to reach saturation with respect to tRNA binding.