. Author manuscript; available in PMC: 2009 Mar 11.

Published in final edited form as: Biochemistry. 2006 Jul 4;45(26):8079–8087. doi: 10.1021/bi060189c

Table 1.

Aminoacylation kinetics for wild-type and mutant Hp ND-AspRS variants.^a

Enzyme	K_M tRNA^Asp (μM)	k_cat tRNA^Asp (sec⁻¹)	k_cat/K_M tRNA^Asp (M⁻¹ sec⁻¹)	K_M tRNA^Asn (μM)	k_cat tRNA^Asn (sec⁻¹)	k_cat/K_M tRNA^Asn (M⁻¹ sec⁻¹)	Specificity Ratio^b
Wild-type	0.77 ± 0.14	0.022 ± 0.001	2.9 × 10⁴	0.83 ± 0.44	0.014 ± 0.001	1.7 × 10⁴	1.7
L81N	0.85 ± 0.22	0.028 ± 0.001	3.3 × 10⁴	1.87 ± 0.24	0.026 ± 0.001	1.4 × 10⁴	2.4
L86M	0.87 ± 0.05	0.021 ± 0.001	2.4 × 10⁴	2.23 ± 0.34	0.015 ± 0.001	0.7 × 10⁴	3.4
L81N/L86M^c	[0.6]	[0.021]	[3.5 × 10⁴]	[1.3]	[0.018]	[1.4 × 10⁴]	[2.5]

Full kinetic analyses were conducted with wild-type ND-AspRS and its mutants. See experimental methods section and Figure 4 for details. All experiments were run at least in triplicate.

The specificity ratio is the k_cat/K_M for tRNA^Asp divided by the k_cat/K_M for tRNA^Asn.

The L81N/L86M mutation did not follow Michaelis-Menten kinetics. The kinetic values presented here were extracted from tRNA concentrations ≤ 4 μM (dashed line, Figure 4D). They are shown in brackets to highlight the fact that they only represent an estimation based on low concentrations of tRNA; at tRNA concentrations ≥ 16 μM, significant inhibition was observed (solid lines, Figure 4D). See text and Figure 4 for further details.