Table 2.
Kinetic analysis of wild-type and mutant GlnRS enzymes
| kcat (s−1) | Km (GLN) (mM) | kcat/Km (s−1M−1) | kobs (s−1) | Kd (GLN) (mM) | |
|---|---|---|---|---|---|
| WT GlnRSa | 3.2 ± 0.5 | 0.26 ± 0.04 | 1.2 × 104 | 28 ± 2 | 1.1 ± 0.04 |
| C229S | 0.7 ± 0.2 (4.6)b | 1.2 ± 0.03 (4.6) | 580 (21) | 20.1 ± 5.1 (1.4) | 7.8 ± 2.1 (7.1) |
| H215N | 2.0 ± 0.1 (1.6) | 0.9 ± 0.2 (3.5) | 2220 (5) | 28.6 ± 1.4 (1.0) | 7.9 ± 0.1 7.2 |
| E257A | 2.3 ± 0.1 (1.4) | 2.0 ± 0.1 (7.7) | 1150 (10) | 25.5 ± 7.5 (1.1) | 9.2 ± 2.7 (8.4) |
| S227A | 4.6 ± 0.3 (0.7) | 3.1 ± 0.7 (11.9) | 1480 (8) | 21.0 ± 4.8 (1.3) | 11.2 ± 0.5 (10.2) |
| D212A | 11.6 ± 4.0 (0.3) | 60 ± 4 (230) | 193 (62) | >122c (<0.2) | >70c (>63) |
| Y211F | 1.2 ± 0.1 (2.7) | 35.5 ± 0.5 (140) | 34 (350) | NDd | ND |
| Q255N | 0.5 ± 0.2 (6.4) | 17.1 ± 3.0 (66) | 30 (400) | ND | ND |
| R30K | 2.6 ± 0.3 (1.2) | 6.3 ± 0.7 (24) | 410 (29) | 2.3 ± 0.3 (12.1) | 5.7 ± 3.0 (5.2) |
a
Values for wild-type GlnRS are taken from ref 26.
b
Numbers in parentheses designate the fold diminution from wild-type for kcat, kcat/Km, and kobs, and the fold elevation from wild-type for Km and Kd.
c
No saturation with glutamine could be observed at concentrations up to 70 mM.
d
Reproducible measurements of kobs and Kd [GLN] could not be made, possibly due to aggregation of the protein.