TABLE 3.
Effects of mutations on kinetic parameters of BoGT6a
| Parametera | Ka | Kb | kcat | Relative kcat |
|---|---|---|---|---|
| mm | mm | s−1 | ||
| UDP-GalNAc substrate | ||||
| Wild type | 0.11 ± 0.02 | 0.69 ± 0.04 | 34 ± 1.5 | 1 |
| N95D | 4.0 ± 1.6 | 3.3 ± 0.58 | 8 ± 0.8 × 10−3 | 2.3 × 10−4 |
| N97D | 0.08 ± 0.01 | 6.4 ± 0.9 | 4.9 ± 0.4 | 0.14 |
| A155M | 0.85 ± 0.06 | 0.27 ± 0.03 | 0.08 ± 0.003 | 2.3 × 10−3 |
| A155Q | 1.35 ± 0.37 | 1.3 ± 0.3 | 0.027 ± 0.003 | 8 × 10−4 |
| E192Q | 0.52 ± 0.21 | 1.7 ± 1.0 | 1.5 ± 0.5 × 10−3 | 4.4 × 10−5 |
| R229A | 0.61 ± 0.06 | 1.7 ± 0.2 | 11.9 ± 0.8 | 0.34 |
| K231A | 0.24 ± 0.04 | 3.4 ± 0.1 | 0.15 ± 0.003 | 3.8 × 10−2 |
| R243A/R244A | 0.23 ± 0.02 | 2.4 ± 0.25 | 3.4 ± 0.17 | 0.1 |
| UDP-Gal substrate | ||||
| Wild type | 0.45 ± 0.04 | 1.35 ± 0.20 | 0.023 ± 0.002 | 6.8 × 10−4 (1)b |
| A155M | 1.27 ± 0.15 | 1.43 ± 0.35 | 0.11 ± 0.007 | 3.2 × 10−3 (4.7) |
| A155Q | 0.98 ± 0.10 | 0.27 ± 0.2 | 0.090 ± 0.004 | 2.6 × 10−3 (3.9) |
a These are apparent values, determined by varying the concentration of one substrate at a fixed concentration (1 mm) of the second. Ka is the apparent Km for UDP-GalNAc or UDP-Gal, and Kb is the apparent Km for 2′-fucosyllactose; kcat (app) is calculated from the apparent Vm determined by varying 2′-fucosyllactose at 1 mm UDP-GalNAc or UDP-Gal.
b Activity relative to that of wild-type enzyme with UDP-Gal as substrate.