TABLE 2.
Steady-state kinetic parameters of wild-type and mutant GmSAT
Reactions were performed as described under “Experimental Procedures.” All parameters are expressed as mean ± S.E. for n = 3. ND, activity was not detected.
| Protein | V/Et | Kmacetyl-CoA | kcat/Kmacetyl-CoA | Kmserine | kcat/Kmserine |
|---|---|---|---|---|---|
| s−1 | μm | m−1 s−1 | μm | m−1 s−1 | |
| Wild type | 70 ± 10 | 321 ± 17 | 218,069 | 617 ± 102 | 113,452 |
| Catalytic site | |||||
| D168A | 0.15 ± 0.05 | 716 ± 182 | 209 | 1,810 ± 292 | 83 |
| D168N | 5.5 ± 0.4 | 244 ± 41 | 22,541 | 1,203 ± 205 | 4,572 |
| H169A | ND | ||||
| H169N | 0.05 ± 0.01 | 330 ± 72 | 152 | 404 ± 54 | 124 |
| H189A | ND | ||||
| H189N | 2.3 ± 0.2 | 2,890 ± 553 | 796 | 3,100 ± 761 | 742 |
| Serine site | |||||
| E177Q | 16 ± 5 | 370 ± 70 | 43,243 | 1,767 ± 387 | 9,055 |
| R203A | ND | ||||
| R203K | 7.8 ± 1.0 | 590 ± 83 | 13,220 | 36,022 ± 5,680 | 217 |
| H204A | 7.9 ± 1.9 | 506 ± 86 | 15,613 | 1,850 ± 358 | 4,270 |
| H204N | 57 ± 8 | 438 ± 41 | 130,137 | 845 ± 279 | 67,456 |
| CoA site | |||||
| A215V | 93 ± 8 | 598 ± 77 | 155,519 | 908 ± 79 | 102,423 |
| K230M | 1.9 ± 0.2 | 5,052 ± 1,030 | 376 | 702 ± 114 | 2,707 |
| A233V | 62 ± 15 | 561 ± 138 | 110,517 | 635 ± 125 | 97,638 |
| T246A | 41 ± 18 | 1,834 ± 656 | 22,356 | 935 ± 237 | 43,850 |
| R253A | 74 ± 9 | 567 ± 146 | 130,511 | 625 ± 76 | 118,400 |