Table 1 |.
Steady-state kinetics of wt-ImGPS in comparison to ImGPS(fS55AzoF), ImGPS(fY39NPY), and ImGPS(fK99mNPK). (See also Figure S4 and Figure S6.)
| PrFAR-dependent HisF activitya | ||||
|---|---|---|---|---|
| protein | state | kcat [min−1] | KmPrFAR [μM] | kcat/Km [M−1 s−1] |
| wt-HisF | 172.6 ± 1.6 | 2.5 ± 0.1 | 11.5 ± 0.5 • 105 | |
| fS55AzoF | E | 48.6 ± 0.9 | 4.8 ± 0.1 | 1.7 ± 0.0 • 105 |
| fS55AzoF | Z | 18.6 ± 0. | 2.4 ± 0.2 | 1.3 ± 0.1 • 105 |
| fY39NPY | caged | 11.3 ± 0.4 | 3.4 ± 0.5 | 0.6 ± 0.1 • 105 |
| fK99mNPK | caged | 112.4 ± 4.7 | 3.2 ± 0.5 | 5.9 ± 0.9 • 105 |
| Glutamine-dependent ImGPS activityb | ||||
| protein | state | kcat [min−1] | KmGln [mM] | kcat/Km [M−1 s−1] |
| wt-ImGPS | 41.4 ± 1.1 | 0.78 ± 0.05 | 8.6 ± 1.1 • 102 | |
| ImGPS(fS55AzoF) | E | 6.0 ± 0.2 | 0.23 ± 0.03 | 4.3 ± 0.6 • 102 |
| ImGPS(fS55AzoF) | Z | 2.6 ± 0.2 | 0.33 ± 0.11 | 1.3 ± 0.4 • 102 |
| ImGPS(fY39NPY) | caged | 3.5 ± 0.2 | 0.25 ± 0.05 | 2.3 ± 0.5 • 102 |
| ImGPS(fK99mNPK) | caged | 7.3 ± 0.4 | 0.41 ± 0.09 | 3.0 ± 0.7 • 102 |
| ProFAR-dependent HisH activity (pH 7.0)c | ||||
| protein | state | kcat [min−1] | KacProFAR [μM] | LRF |
| wt-ImGPS | 16.8 ± 0.3 | 32.8 ± 1.2 | ||
| ImGPS(fS55AzoF) | E | 5.4 ± 0.4 | 19.5 ± 3.8 | 2.3 |
| Z | 2.3 ± 0.2 | 35.1 ± 5.0 | ||
| ImGPS(fY39NPY) | caged | 1.2 ± 0.1 | 32.3 ± 4.2 | 5.9 |
| decaged | 7.0 ± 0.5 | 28.7 ± 4.2 | ||
| ImGPS(fK99mNPK) | caged | 3.5 ± 0.3 | 19.6 ± 3.7 | 4.0 |
| decaged | 14.0 ± 0.9 | 32.1 ± 3.2 | ||
a,b
Values ± s.e. for kcat and Km were determined by Michaelis-Menten fitting of the mean ± s.e.m. for at least two technical replicates. Values ± s.e. for kcat/Km were calculated according to the Gaussian law of error propagation.
c
Values ± s.e. for kcat and KacProFAR were determined by fitting the mean ± s.e.m. for at least two technical replicates to a hyperbolic function. LRF = kcatE/kcatZ or kcatdecaged/kcatcaged