TABLE 3.
Specific activities of wild-type 103S_EGCase I and its mutants
Representative HPLC chromatograms of the specific activity assay are presented in supplemental Fig. S4.
| 103S_EGCase I mutant | GM1 |
LacCer |
Interacting sugar units | ||
|---|---|---|---|---|---|
| Specific activity | Relative activity | Specific activity | Relative activity | ||
| nmol/min/μg | % | nmol/min/μg | % | ||
| WT | 7.59 ± 0.23 | 100 | 1.92 ± 0.17 | 0 | |
| K61A | 0 | 0 | 0 | 0 | Glc (O3), Gala (O4, O6) |
| D62A | 5.46 ± 0.09 | 71.9 | 1.61 ± 0.03 | 83.9 | GalNAc (O4) |
| H131A | 0 | 0 | 0 | 0 | Glc (O3) |
| D133A | 0 | 0 | 0 | 0 | Glc (O2, O3) |
| N213A | 0 | 0 | 0 | 0 | Glc (O2) |
| N265A | 0 | 0 | 0.04 ± 0.02 | 2.1 | Sphingosine(OH) |
| Q298A | 0 | 0 | 0 | 0 | Glc (O6), Fatty acid (O) |
| Y302A | 0 | 0 | 0 | 0 | Glc (O6), Gala (O2) |
| E339S | 0 | 0 | 0 | 0 | Glc(O2) |
| D342A | 0 | 0 | 0 | 0 | NeuAc (O4) |
| W365A | 0 | 0 | 0 | 0 | Gala (O2) |
a The inner Gal unit in the GM1 sugar chain.