Table 1.
Influence of N-terminal prodomain and intersubunit linker on caspase-6 activity
| Caspase-6 Construct | Pro domain |
Linker | Expected MW |
Observed MW |
Km (μM) | kcat (s−1) | Observed MW SEC (kDa) |
Observed MW AUC (kDa) |
|---|---|---|---|---|---|---|---|---|
|
Yes | Yes | 34180 | 34179 | 43 ± 0.51 | 0.04 ± 0.001 | 66 | 85 |
|
Yes | Yes | 21892 12350 |
21894 12351 |
67 ± 3.2 | 0.84 ± 0.07 | 78 | NA |
|
Yes | No | 20265 12109 |
20264 12109 |
51 ± 3.5 | 0.82 ± 0.03 | 34 | 64 |
|
No | Yes | 19624 12350 |
19624 12351 |
66 ± 7 | 0.81 ± 0.01 | 56 | NA |
|
No | No | 18066 12350 |
18068 12351 |
45 ± 13 | 0.92 ± 0.14 | 35 | 64 |
UC - uncleavable. CT - constitutively two-chain. ΔN lacks the N-terminal prodomain. Expected molecular weight (MW) is calculated based on the gene sequence in the expression construct. Observed MW is based on mass spectrometric analysis. Kinetic parameters are based on substrate titrations measured from independent duplicate dilutions of substrate on three days. SEC Size Exclusion Chromatography. AUC Analytical Ultracentrifugation. NA Not Attempted.