Table 1.
Steady State Kinetics with Acs Mutations
| Enzyme | KM ATP (µM) |
kcat ATP (s−1) |
kcat/KM ATP (µM−1 s−1) |
KM CoA (µM) |
kcat CoA (s−1) |
kcat/KM CoA (µM−1 s−1) |
|---|---|---|---|---|---|---|
| Wild-type | 77.1 ± 13 | 100.6 ± 2.6 | 1.3 ± .2 | 50 ± 4.5 | 95.1 ± 1.9 | 1.9 ± .2 |
| Mutations that affect the adenylation half-reaction | ||||||
| K609Aa | -- | -- | -- | -- | -- | -- |
| Mutations of residues that interact with CoA or stabilize the thioester-forming conformation | ||||||
| R194A | 28.7 ± 2.2 | 39.9 ± 0.5 | 1.4 ± .1 | 141.7 ± 17.4 | 41.1 ± 0.7 | 0.3 ± .04 |
| R194E | 37.3 ± 5.7 | 41.7 ± 1.3 | 1.1 ± .2 | 107.2 ± 11.9 | 40.0 ± 1.5 | 0.4 ± .05 |
| R584A | 38.1 ± 5.5 | 46.7 ± 1.2 | 1.2 ± .2 | 358.3 ± 60.6 | 41.5 ± 1.1 | 0.1 ± .02 |
| R584E | 26.5 ± 2.3 | 36.4 ± 0.6 | 1.4 ± .1 | 426.0 ± 39.0 | 40.8 ± 0.8 | 0.09 ± .009 |
| R526A | 22.4 ± 3.5 | 37.4 ± 1.3 | 1.7 ± .3 | 205.0 ± 31.2 | 40.5 ± 0.6 | 0.2 ± .03 |
| Mutations that border the pantetheine binding tunnel | ||||||
| A357V | 23.8 ± 2.7 | 38.9 ± 0.5 | 1.6 ± .2 | 133.0 ± 18.0 | 35.0 ± 0.8 | 0.3 ± .04 |
| G524S | 63.8 ± 7.3 | 49.8 ± 1.6 | 0.8 ± .09 | 448.0 ± 90 | 43.1 ± 1.1 | 0.1 ± .02 |
| G524La | -- | -- | -- | -- | -- | -- |
| Mutations in the hinge residue, Asp517 | ||||||
| D517P | 44.0 ± 3.2 | 40.9 ± .7 | 0.9 ± .07 | 527.8 ± 60 | 43.8 ± 1.5 | 0.08 ± .009 |
| D517G | 243.0 ± 13.8 | 42.8 ± 0.6 | 0.2 ± .01 | 228.0 ± 31.7 | 42.4 ± 0.7 | 0.2 ± .03 |
a
No detectable activity was observed for K609A and G524L.