Table 1.
Wild-type and mutant Pus7 single turnover rate constants and dissociation constants for CDC8
| PUS7 | kobs (s−1)* | KD,app1 (nM)† |
| WT | 9.9 ± 1.0 × 10−1 | 75 ± 15 |
| K61A | 2.6 ± 0.1 × 10−2 | 180 ± 40 |
| F67A | 4.6 ± 0.2 × 10−3 | 480 ± 50 |
| E71A | 5.2 ± 0.3 × 10−3 | 210 ± 50 |
| H161A | 6.9 ± 0.9 × 10−1‡ | 170 ± 40 |
| D256A | N.A.§ | 60 ± 15 |
| N305A | 4.0 ± 0.1 × 10−4 | 230 ± 60 |
| F307A | 1.3 ± 0.1 × 10−5 | 340 ± 170 |
| F307Y | 2.6 ± 0.1 × 10−3 | N.D.¶ |
| ΔID1 | 3.8 ± 0.6 × 10−1‡ | 160 ± 40 |
*kobs values were determined by tritium release assays using 2 μM PUS7 and <100 nM CDC8 substrate. At least three replicate curves were collected for each mutant.
†KD,app1 values were determined by EMSAs (n ≥ 2) using Pus7 containing the catalytically inactive D256A mutation in addition to the mutation indicated in the first column.
‡kobs value determined using 10 μM Pus7 and <100 nM CDC8 substrate.
§N.A., no activity.
¶N.D., not determined.