Table 1. Them2-catalysed acyl-CoA hydrolysis: steady-state kinetic constants and characteristics.
Reactions were carried out at 37°C in 50 mM KCl, pH 7.5, unless otherwise specified. As determined for Them2 using myristoyl-CoA as the substrate, the optimal pH was ≥7.2 and the optimal range for ionic strength was 80–160 mM for both NaCl and KCl. Values of Km and Vmax were determined by fitting the experimental data to the Michaelis–Menten equation as described in the text. For the determination of kinetic constants, each data point (V0) in the saturation curve represents the average of duplicate determinations. The kinetic constants reported for an experiment represent non-linear regression fits to these data. Protein concentrations were determined using molar absorbance values of Them2. Them2 concentrations measured using the Bradford method were 3-fold lower. Therefore, if used for calculating steady-kinetic constants, protein concentrations measured by the Bradford method would increase Vmax, kcat and kcat/Km 3-fold. Values of kcat were calculated assuming that Them2 was tetrameric in solution. We observed slight differences in activity among Them2 preparations, which may have been due to the storage time of the protein and times required for mixing of reagents. In order to estimate these effects, the kinetic constants were determined on three separate occasions for myristoyl-CoA at 37°C. This revealed a variability (1 S.D.) of 8% for Km and 13% for Vmax.
| Substrate | Km (µM) | Vmax (nmol/min per mg) | kcat (s−1) | kcat/Km (M−1 · s−1) |
|---|---|---|---|---|
| Fatty acyl-CoAs | ||||
| Acetyl-CoA | Inactive | |||
| 2-Butenoyl-CoA | Inactive | |||
| Hexanoyl-CoA (C6:0) | 138.2 | 29.8 | 3.0 × 10−2 | 2.2 × 102 |
| Decanoyl-CoA (C10:0) | 47.2 | 54.3 | 5.5 × 10−2 | 1.2 × 103 |
| Lauroyl-CoA (C12:0) | 27.5 | 37.7 | 3.8 × 10−2 | 1.4 × 103 |
| Myristoyl-CoA (C14:0) | 15.5 | 46.9 | 4.7 × 10−2 | 3.1 × 103 |
| 25°C | 7.3 | 30.4 | 3.1 × 10−2 | 4.4 × 103 |
| 50°C | 21.4 | 61.4 | 6.2 × 10−2 | 2.9 × 103 |
| Phosphatidylcholine (4 mM)* | 22.8 | 32.2 | 3.3 × 10−2 | 1.4 × 103 |
| StarD2/Them2,0.5 | 10.6 | 68.1 | 6.9 × 10−2 | 6.5 × 103 |
| StarD2/Them2,1.0 | 13.6 | 84.6 | 8.6 × 10−2 | 6.3 × 103 |
| Palmitoyl-CoA (C16:0) | 10.0 | 38.1 | 3.8 × 10−2 | 3.8 × 103 |
| StarD2/Them2,0.5 | 18.0 | 56.8 | 5.7 × 10−2 | 3.2 × 103 |
| StarD2/Them2,1.0 | 15.9 | 65.2 | 6.6 × 10−3 | 4.2 × 103 |
| Oleoyl-CoA (C18:1,n−9,cis) | 27.4 | 60.4 | 6.1 × 10−2 | 2.2 × 103 |
| Other acyl-CoAs | ||||
| β-Hydroxybutyryl-CoA | 162.8 | 45.4 | 4.6 × 10−2 | 2.8 × 102 |
| 3-Hydroxy-3-methylglutaryl-CoA | 305.7 | 6.9 | 7.0 × 10−3 | 2.3 × 101 |
| Malonyl-CoA | 142.3 | 14.0 | 1.4 × 10−2 | 1.0 × 102 |
| Phenylacetyl-CoA | 191.6 | 119.8 | 1.2 × 10−1 | 6.3 × 102 |
Determined in the presence of small unilamellar vesicles composed of pure phosphatidylcholine at 4 mM total phospholipid concentration.