Table 3.
Kinetic Parameters for wild-type rS-HPCDH3 and R211A and K214A with various substratesa
| Enzyme | Km (mM) | Change in Km (x-fold) | kcat (s-1) | Change in kcat (x-fold) | kcat/Km (M-1 s-1) |
|---|---|---|---|---|---|
| Substrate: S-HPC | |||||
| wild-type | 0.031 ± 0.001 | 1.0 | 25 ± 0.2 | 1.0 | 7.9 × 105 |
| R211A | 1.6 ± 0.1 | 49 | 16 ± 0.4 | 0.64 | 1.0 × 104 |
| K214A | 2.3 ± 0.1 | 73 | 5.8 ± 0.1 | 0.23 | 2.5 × 103 |
| Substrate: 2-KPC | |||||
| wild-type | 0.28 ± 0.01 | 1.0 | 11 ± 0.2 | 1.0 | 3.9 × 104 |
| R211A | 11 ± 0.4 | 41 | 8.6 ± 0.2 | 0.80 | 7.5 × 102 |
| K214A | 12 ± 0. 9 | 43 | 9.6 ± 0.3 | 0.89 | 8.1 × 102 |
| Substrate: 2-butanone | |||||
| wild-type | 120 ± 5 | 1.0 | 0.044 ± 0.001 | 1.0 | 3.7 × 102 |
| R211A | 180 ± 30 | 1.5 | 0.072 ± 0.05 | 1.6 | 4.0 × 102 |
| K214A | 72 ± 6 | 0.61 | 0.039 ± 0.001 | 0.90 | 5.4 × 102 |
| Substrate: 2-propanol | |||||
| wild-type | 1400 ± 60 | 1.0 | 2.0 ± 0.5 | 1.0 | 1.4 × 103 |
| R211A | 720 ± 40 | 0.51 | 1.7 ± 0.03 | 0.85 | 2.4 × 103 |
| K214A | 950 ± 20 | 0.67 | 1.7 ± 0.2 | 0.86 | 1.8 × 103 |
Assay for S-HPC oxidation contained 0.20 μg of wild-type rS-HPCDH3, or 4.0 μg of R211A and K214A while assay for 2-KPC reduction contained 0.20 μg of wild-type rS-HPCDH3, 5.0 μg of R211A and 2.0 μg of K214A. Assay for 2-butanone reduction contained 197 μg of enzyme while assay for 2-propanol oxidation contained 8.0 μg of enzyme. Apparent kcat and Km values are reported as means ± standard deviations. All other values are reported as means only. All assays were performed in triplicates at 30 °C with fixed concentrations of NAD+ (10 mM) or NADH (0.17 mM). Apparent kinetic constants were determined by fitting experimental data to the standard form of the Michaelis-Menten equation.