Table 2.
Proteolytic kinetics of the CBS mutants under native conditions and pulse proteolysis in a urea gradient
| Proteolysis under native conditions | Pulse proteolysis | ||
|---|---|---|---|
| Protein | kp (106. M−1.min−1) | B (%) | Cm [M] |
| Wild-type | 0.30 ± 0.04 | 24.4 ± 1.7 | 3.05 ± 0.03 |
| p.H65R | 38.4 ± 5.1 | 8.2 ± 2.1 | N.D |
| p.A114V | 2.2 ± 0.4 | 24.1 ± 2.5 | 2.84 ± 0.04* |
| p.T191M | 44.3 ± 8.8 | 22.9 ± 3.0 | 2.4** |
| p.I278T | <2, 64.5>*** | 19.5 ± 1.2 | 2.42 ± 0.01 |
| p.E302K | 5.0 ± 0.6 | 0 ± 3.3 | N.D |
| p.G307S | 4.6 ± 0.4 | 0 ± 0.1 | N.D |
| p.R369C | 24.0 ± 3.5 | 23.4 ± 1.9 | 2.59 ± 0.09 |
| p.R439Q | 0.02 ± 0.01 | 25.7 ± 9.6 | 3.49 ± 0.04 |
| p.D444N | 0.03 ± 0.01 | 28.4 ± 6.2 | 3.32 ± 0.04 |
k p, B and c m values were determined by nonlinear data fitting from four different measurements. Representative gels with corresponding plots are shown in the Supplementary materials.
*The determination of c m for p.A114V neglected the cleavage of partially folded forms.
**Values were determined by visual inspection because these data could not be evaluated by nonlinear data fitting.
***k p could not be exactly determined due to low reproducibility at lower concentrations of thermolysin.
N.D. not determined due to the rapid proteolysis in the absence of urea.