TABLE 1.
Kinetic parameters of reconstituted HCM subunits HcmA (wild-type Ile90 and mutant I90V) and wild-type HcmB incubated with various acyl-CoA substrates at pH 6.6 and 30 °C (Fig. 5).
| Subunit and substrate | Vmaxa | Km | kcat | kcat/Km | Vmax/Km |
|---|---|---|---|---|---|
| nmol min−1mg−1 | μm | min−1 | mm−1 min−1 | liters min−1mg−1 | |
| HcmA Ile90 | |||||
| (S)-3-Hydroxybutyryl-CoA | 140 ± 5.6 (100)b | 128 ± 22 | 12 ± 0.5 | 90 ± 16 | 1,080 ± 190 |
| 2-Hydroxyisobutyryl-CoA | 36 ± 2.8 (26) | 104 ± 25 | 3.0 ± 0.2 | 29 ± 7.3 | 350 ± 87 |
| (R)-3-Hydroxybutyryl-CoA | 2.4 ± 0.15 (2) | 1,660 ± 367 | 0.20 ± 0.01 | 0.12 ± 0.03 | 1.4 ± 0.3 |
| Butyryl-CoA | 2.4 ± 0.26 (2) | 3,340 ± 880 | 0.20 ± 0.02 | 0.06 ± 0.02 | 0.7 ± 0.2 |
| Isobutyryl-CoA | 0.67 ± 0.03 (0.5) | 550 ± 140 | 0.06 ± 0.003 | 0.10 ± 0.03 | 1.2 ± 0.3 |
| HcmA I90V | |||||
| (S)-3-Hydroxybutyryl-CoA | 24 ± 1.1 (17) | 1,760 ± 240 | 2.0 ± 0.1 | 1.1 ± 0.16 | 14 ± 2.0 |
| 2-Hydroxyisobutyryl-CoA | 15 ± 1.1 (11) | 1,840 ± 370 | 1.2 ± 0.1 | 0.7 ± 0.14 | 8.1 ± 1.7 |
a Activities obtained with the wild-type HcmAB for methylmalonyl- and succinyl-CoA and with the HcmA I90V mutant reconstituted with wild-type HcmB for all acyl-CoA esters tested, except for (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA, were below detection limit (<0.01 nmol min−1 mg−1).
b For calculating relative activities, Vmax obtained with wild-type HcmAB for (S)-3-hydroxybutyryl-CoA was set to 100%.