Table 2.
Summary of analytical ultracentrifugation and kinetic data
| Protein | Kd (μM)b (monomer- dimer) | Kd (μM)b (dimer- tetramer) | kcat (s−1) | Km (mM) (for pyruvate) | kcat/Km (s−1M−1) |
|---|---|---|---|---|---|
| Full-length SaPC a | |||||
| Wild-type | 0.75±0.06 | 0.008±0.0006 | 20.2±1.4 | 3.4±0.8 | 5900±1400 |
| R54E | 0.53±0.03 | 0.003±0.0001 | N.A.c | N.A. | N.A. |
| F403A | 0.45±0.04 | 0.011±0.0006 | 1.3±0.1 | 1.4±0.4 | 930±270 |
| K442E | 0.82±0.09 | 840±190 | N.A. | N.A. | N.A. |
| EcBC chimera | 2.31±0.33 | 0.28±0.03 | 1.1±0.1 | 0.34±0.02 | 3300±200 |
| ScBC chimera | 0.56±0.06 | 68±11 | N.A. | N.A. | N.A. |
| BC domain of SaPC a | |||||
| Wild-type | 48±4 | — | N.A. | N.A. | N.A. |
| R54E | 195±7 | — | N.A. | N.A. | N.A. |
| F403A | 130±6 | — | N.A. | N.A. | N.A. |
| K442E | 86±8 | — | N.A. | N.A. | N.A. |
a
The protein concentrations for full-length SaPC were 0.4, 1.5 and 7.4 μM (based on monomer) in the AUC experiment, and those for the BC domain were 1, 4, and 10 μM.
b
For full-length SaPC, the Kd values were the best-fit results based on a rapid monomer-dimer-tetramer association model, and the local root-mean-square deviation (rmsd) was from 0.0041 to 0.0210. For BC domain alone, a rapid monomer-dimer association model was used, and the local rmsd was from 0.0037 to 0.0070.
c
N.A. – No activity was observed under the condition tested.