Table 2. Activity and UDP-glucose binding of single- and double-mutants of LppGT compared with wild-type enzyme.
The Kd for UDP-glucose was determined by fitting fluorescence intensity data, obtained by tryptophan fluorescence experiments, against free UDP-glucose concentration (Figure 3A). The activity and Kd experiments results represent means ± S.D. for three independent experiments. The percentage of surviving and intact HeLa cells was determined after 48 h; intact cells were not apoptotic and rounded and represent the mean of two independent experiments. nd, not detectable; –, not performed.
| LppGT varient | % activity | Kd (nM) | % surviving cells | % intact cells |
|---|---|---|---|---|
| Wild-type | 100 | 100±29 | 33 | 10 |
| D246A | 48±3 | 400±80 | 38 | 18 |
| D248A | 92±3 | 990±90 | 36 | 18 |
| D246A/D248A double-mutant | nd | 830±100 | 100 | 95 |
| N293A | nd | 72±30 | 93 | 90 |
| E445A | 86±7 | 41±25 | – | – |
| E446A | 78±10 | 130±40 | – | – |
| Y454A | 43±2 | 60±30 | – | – |
| N499A | nd | 90±20 | 110 | 110 |
| S519A | 87±4 | 100±43 | – | – |
| GST | – | – | 305 | 300 |
| Buffer | – | – | 247 | 245 |