Table 1.
The apparent rate constants for the wild-type and mutant thioesterase catalysis of multiple and single turnover hydrolysis of 4-HB-CoA as measured using stopped-flow UV-vis absorbance spectroscopy (sf) and rapid quench (rq) techniques. All entries that are not designated were measured using stopped-flow UV-vis absorbance spectroscopy. Biphasic time courses were fitted to a double exponential equation and single phase time courses were fitted to a single exponential equation.
| Thioesterase | Multiple Turnover | Single Turnover | ||
|---|---|---|---|---|
| Single phase | Double phase | |||
| k (s−1) | Fast phase k (s−1) | Slow phase k2 (s−1) | k (s−1) | |
| Wild-type | 40 ± 2 (sf)a 100 ± 30 (rq) |
6.9 ± 0.4 (sf)a 8.7 ± 0.3 (rq) |
28.7 ± 0.1 (sf) 36 ± 2 (rq) |
|
| E73D | 0.0614 ± 0.0002 (sf) 0.0774 ± 0.001 (rq) |
-------- -------- |
-------- -------- |
0.774 ± 0.001 -------- |
| T77A | -------- -------- |
19.7 ± 0.2 (sf) 47 ± 8 (rq) |
0.49 ± 0.01(sf) 1.38 ± 0.06 (rq) |
17.0 ± 0.1 (sf) 24 ± 2 (rq) |
| T77S | -------- | 18.9 ± 0.2 | 1.208 ± 0.006 | 19 s−1 |
| T77V | 0.152 ± 0.002 | -------- | -------- | -------- |
| E73D/T77A | 0.705 ± 0.001 | -------- | -------- | -------- |
| E73D/T77S | 0.0619 ± 0.0001 | -------- | -------- | -------- |
| Q58A | 0.0842 ± 0.0001 | -------- | -------- | -------- |
| Q58E | 0.288 ± 0.001 | -------- | -------- | -------- |
| E78A | 0.175 ± 0.001 | -------- | -------- | -------- |
| H64A | 0.0139 ± 0.0001 | -------- | -------- | -------- |
| H64Q | 0.0691 ± 0.0001 | -------- | -------- | -------- |
| D31A | 0.0120 ± 0.0001 | -------- | -------- | -------- |
| D31N | 0.420 ± 0.002 | -------- | -------- | -------- |
a
These error limits are based on the standard deviation of three individual measurements. All of the other error limits shown are based on the fit to a single representative data set.