Table 1.
Kinetic parameters of recombinant IPTs
| Enzyme | Substrate | Km, μM | Vmax, nmol·min−1·mg protein−1 | kcat, min−1 | kcat/Km, min−1/M−1 | Sp. ratio |
|---|---|---|---|---|---|---|
| Tzs | DMAPP | 7.9 ± 0.6 | 119.0 ± 10.1 | 3.2 | 4.1 × 105 | 2.3 |
| HMBDP | 8.2 ± 0.4 | 55.0 ± 1.3 | 1.5 | 1.8 × 105 | ||
| AMP | 0.035 ± 0.005 | |||||
| TzsD173G | DMAPP | 2.6 ± 0.3 | 50.8 ± 1.4 | 1.4 | 5.4 × 105 | 208 |
| HMBDP | 22.6 ± 1.0 | 2.2 ± 0.1 | 5.8 × 10−2 | 2.6 × 103 | ||
| TzsE210N | DMAPP | 4.2 ± 0.1 | 51.4 ± 0.7 | 1.4 | 3.3 × 105 | 15.0 |
| HMBDP | 26.1 ± 1.1 | 22.5 ± 0.7 | 0.6 | 2.2 × 104 | ||
| TzsY211T | DMAPP | 35.4 ± 2.1 | 4.7 ± 0.2 | 0.13 | 3.6 × 103 | 9.0 |
| HMBDP | 33.5 ± 1.5 | 0.5 ± 0.1 | 1.4 × 10−2 | 4.0 × 102 | ||
| TzsE213Q | DMAPP | 7.5 ± 0.3 | 72.5 ± 2.6 | 2.0 | 2.6 × 105 | 5.1 |
| HMBDP | 27.2 ± 0.8 | 51.5 ± 1.0 | 1.4 | 5.1 × 104 | ||
| TzsH214L | DMAPP | 10.8 ± 0.9 | 199.0 ± 16.0 | 5.4 | 5.0 × 105 | 1163 |
| HMBDP | 80.5 ± 9.6 | 1.3 ± 0.1 | 3.5 × 10−2 | 4.3 × 102 |
The Km values for DMAPP and HMBDP were measured by using a nonradioisotope assay (30) with AMP (50 μM), and for AMP with DMAPP (100 μM). Means ± SD (n = 3). Sp. ratio, specificity ratio: the kcat/Km for DMAPP divided by the kcat/Km for HMBDP.