Table 3.

Pre-steady state kinetic analysis of the tryptophan activation reaction by stopped-flow kinetics ^a.

$\text{E}+\text{Trp}+\text{ATP}\stackrel{k_{\text{f}1}}{\to }\text{E Trp-AMP}\stackrel{k_{\text{f}2}}{\to }\text{E}+\text{Trp-AMP}$

TrpRS	KATP (mM)	kf1 (s−1)d
Wild type	2.0 (± 0.3) b,c	13 (± 0.8)
R162A	115 (± 29)	0.05 (± 0.007)
R162A/A352K	2.8 (± 1.2)	0.01 (± 0.001)

^aAt room temperature (23 °C) and pH 8.0.

^bStandard errors for curve fitting of 3 repeated experiments are indicated in parentheses.

^cThe K_M for ATP is much higher than the K_d measured by ITC. This relationship between K_M and K_d is seen for many enzymes and, in this particular instance, is likely because, in the kinetic studies, the conversion of bound ATP to the Trp-AMP product occurs more rapidly than dissociation of free ATP from the enzyme. This conversion, in effect, raises sharply the apparent dissociation rate constant, giving rise to K_M that is much higher than K_d (Cantor and Schimmel, 1980). In addition, for reasons of technical convenience, conditions of the two experiments were some what different (e.g. pH 7.5 for ITC verses pH 8.0 for this kinetic study).

^dThe k_f1 values are calculated based on K_M of 2.5 μM for tryptophan (Ewalt et al., 2005).