TABLE 3.
Kinetic analysis of XdINV mutants
The kcat values were calculated assuming a protein molecular mass of 66 kDa (unglycosylated monomer). The ± sign refers to standard error curve fit using the kinetic module of SigmaPlot version 12. The mutants D80A, N107S, E303A, E334S/E334V/E334N, Y659STOP gave no activity using sucrose. WT, wild type; ND, not detected; blank, not evaluated.
| XdINV variant |
Km (mm) |
kcat (s−1) |
kcat/Km (mm−1 s−1) |
||||||
|---|---|---|---|---|---|---|---|---|---|
| Sucrose | Raffinose | Nystose | Sucrose | Raffinose | Nystose | Sucrose | Raffinose | Nystose | |
| WT | 1.9 ± 0.3 | 7.7 ± 1.6 | 21.7 ± 3.6 | 911 ± 47 | 291 ± 51 | 148 ± 25 | 479 ± 68 | 56 ± 10 | 72 ± 12 |
| N58S | 6.1 ± 1.7 | 711 ± 200 | 117 ± 33 | ||||||
| E334Q | 3.6 ± 0.6 | 15.5 ± 3.4 | 1.9 ± 0.4 | 91 ± 15 | 7 ± 1 | 46 ± 10 | 25 ± 4 | 0.4 ± 0.1 | 23 ± 5 |
| Q341N | 1.8 ± 0.4 | 8.4 ± 2.0 | 30.6 ± 6.9 | 377 ± 76 | 171 ± 40 | 71 ± 16 | 212 ± 43 | 20 ± 5 | 2 ± 1 |
| N342S | 7.3 ± 1.3 | 2.9 ± 0.7 | 12.7 ± 2.1 | 1070 ± 189 | 165 ± 41 | 65 ± 11 | 147 ± 26 | 58 ± 14 | 5 ± 1 |
| H343A | 8.3 ± 1.5 | 7.5 ± 1.5 | ND | 105 ± 19 | 32 ± 6 | ND | 13 ± 2 | 4 ± 1 | ND |
| H343T | 12.6 ± 3.3 | 12.9 ± 3.5 | ND | 855 ± 223 | 101 ± 27 | ND | 68 ± 18 | 8 ± 2 | ND |
| N471S | 3.4 ± 0.9 | 1151 ± 307 | 339 ± 91 | ||||||