Table 1.
Effect of CPS mutations on the ability of transformants to support the growth of the CPS-disrupted strain LPL26 and on CP synthesis activity in crude extracts prepared from transformants.
| Mutant | Generation time, (h)
|
Specific activity
|
||
|---|---|---|---|---|
| 30°C | 37°C | − Gly | + Gly | |
| Wild-type CPS | 4.9 | 5.7 | 1.05 | 3.35 |
| K773A | 5.5 | 4.7 | 1.23 | 5.12 |
| E780A | u | u | u | u |
| E780D | u | u | u | u |
| E780Q | u | u | u | u |
| E780H | u | u | u | u |
| D782A | u | u | u | u |
| D782E | 4.7 | 6.0 | 0.62 | 2.12 |
| D782N | 33.5 | 96.3 | 0.16 | 0.26 |
| D784A | u | u | u | u |
| D784E | 4.8 | 4.8 | 0.27 | 0.57 |
| D784N | u* | u* | 0.19 | 0.46 |
| D784Q | 43.3 | 91.5 | u | u |
| E317A | u | u | u | u |
| N319A | u | u | u | u |
Glycine activation indicates conformational coupling between the synthetase and glutamine amidotransferase subunits of CPS, with glycine binding to the latter as a glutamine analog (44); the presence of such coupling serves as one indication that the mutation in CPS has not resulted in a gross conformational change. Specific activity is nmol CP synthesized/min per unit CPS protein, with amount of CPS protein determined by densitometry of Western blots. u, Below the assay detection limit; u*, D784N failed to grow in liquid media but showed essentially wild-type growth on solid medium.