Table 1. Steady State Aminoacylation Parameters.
| kcat (s–1) | KM [tRNA] (μM) | kcat/KM [tRNA] (s–1 M–1) | KM [GLU] (mM) | kcat/KM [GLU] (s–1 M–1) | ||
|---|---|---|---|---|---|---|
| E. coli GlnRSa | tRNAGLN | 0.046 ± 0.013 | 19 ± 3 | 2.4 × 103 | >750 | 9.5 × 10–4 |
| C229R GlnRSb | tRNAGLN | 3.2 ± 0.1 x10–4 | 240 ± 10 | 1.3 × 10–3 | ||
| S1 GlnRSb | tRNAGLN | 5.0 ± 0.6 x10–3 | 230 ± 17 | 2.2 × 10–2 | ||
| L1L2b | tRNAGLN | 0.09 ± 0.02 | 7.6 ± 3.0 | 1.2 × 104 | 5.8 ± 0.5 | 15.5 |
| L1L2 W256Yc | tRNAGLN | 0.10 ± 0.04 | 4.8 ± 0.2 | 2.1 × 104 | 2.6 ± 1.0 | 38.5 |
| L1L2 W256Yd | Q/E tRNA1 | 0.014 ± 0.01 | 1.2 ± 0.4 | 1.2 × 104 | 14 ± 7 | 1.0 |
| L1L2 W256Y | Q/E tRNA2 | 0.05 ± 0.01 | 1.4 ± 0.7 | 3.6 × 104 | 53 ± 22 | 0.9 |
| L1L2 W256Y | Q/E tRNA3 | 0.32 ± 0.05 | 2.4 ± 0.8 | 1.3 × 105 | 1.9 ± 0.2 | 168.4 |
| L1L2 W256Y | Q/E tRNA4 | 0.069 ± 0.01 | 0.74 ± 0.07 | 9.3 × 104 | 3.3 ± 0.6 | 20.9 |
| L1L2 R237D/R238E/W256Y | Q/E tRNA1 | 0.092 ± 0.01 | 3.4 ± 0.7 | 2.7 × 104 | 12 ± 5 | 7.7 |
| L1L2 R237D/R238E/W256Y | Q/E tRNA4 | 0.29 ± 0.05 | 3.4 ± 0.5 | 8.5 × 104 | 3.0 ± 0.7 | 97.7 |
| benchmark enzymes | ||||||
| E. coli GlnRS + GLNa | tRNAGLN | 3.2 ± 0.5 | 0.31 ± 0.09 | 1.0 × 107 | 0.26 ± 0.04a | 1.2 × 104 |
| M. thermautotrophicus GluRSND(c) | tRNAGLN | 0.12 ± 0.01 | 0.038 ± 0.010 | 3.2 × 106 | 6.2 ± 0.6 | 19.4 |
| S. cerevisiae GluRSD(e) | tRNAGLU | 1.6 ± 0.6 | 0.16 ± 0.03 | 1.0 × 107 | 39 ± 14 | 41.0 |
a
Reported in ref (25). All values in this table reflect glutamylation except for the benchmark represented by E. coli GlnRS for its cognate glutaminylation reaction (E. coli GlnRS + GLN). The value 0.26 ± 0.04 represents KM[GLN]. No activity with GLN as a substrate is detectable for S1 GlnRS or for any of the L1L2 hybrids.
b
Reported in ref (26). S1 GlnRS includes the following mutations: C229R/Q255I/S227A/F233Y. S1L1L2 GlnRS adds the following mutations: T214A/H215C/C216P/S218V/A220S/L221I/I224V/L231T/V243I/L244I/D245E/N246A/I247L/T248G/P250R/ΔV251/H252K/R254Y/Y256W
c
Reported in ref (27)
d
Sequences of hybrid Q/E tRNAs are provided in Figure 2 and the Supporting Information.
e
Reported in ref (32).