Table 2. Kinetics of GTP Hydrolysis by WT and E37A/C66A/C67A YjiAa.
| metal bound | kcat (s–1) | Km (M) | kcat/Km(M–1 s–1) | |
|---|---|---|---|---|
| WT | apo | (6 ± 2) × 10–3 | (5 ± 3) × 10–5 | 14 ± 9 |
| Co(II) | (5 ± 2) × 10–4 | (2.3 ± 0.7) × 10–4 | 2.3 ± 0.8 | |
| Ni(II) | WHb | |||
| Zn(II) | NHc | |||
| E37A/C66A/C67A | apo | (5 ± 1) × 10–4 | (1.0 ± 0.7) × 10–4 | 6 ± 3 |
| Co(II) | (1.1 ± 0.1) × 10–3 | (1.7 ± 0.1) × 10–5 | 78 ± 9 | |
| Ni(II) | (6 ± 1) × 10–4 | (3 ± 1) × 10–4 | 2 ± 1 | |
| Zn(II) | NHc | |||
a
All GTPase assays were conducted with 0.5–2 μM WT or E37A/C66A/C67A YjiA in protein buffer supplemented with 5 mM MgCl2. Samples containing metal were preincubated with either zinc, cobalt, or nickel overnight at 4 °C in an anaerobic glovebox. The amount of released phosphate was detected using a modified Malachite Green assay. The data listed are average values from at least three independent experiments.
b
Weak hydrolysis (see the text for details).
c
No measurable hydrolysis (see the text for details).