TABLE 2.
Kinetic parameters of the wild-type and the heterologously expressed Ffase variants, including the residue Ser or Leu196a
| Substrate | Variant | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) |
|---|---|---|---|---|
| Sucrose | Wt | 7.5 ± 1 | 185 ± 9 | 24.6 |
| Ser196 | 8.2 ± 1.4 | 173.3 ± 16 | 21.1 | |
| Leu196 | 32.9 ± 5.6 | 0.93 ± 0.06 | 0.028 | |
| 1-Kestose | Wt | 2.3 ± 0.6 | 55.3 ± 5 | 24 |
| Ser196 | 2.6 ± 0.7 | 51 ± 5 | 19.6 | |
| Leu196 | 22.6 ± 3.6 | 0.2 ± 0.006 | 0.009 | |
| Nystose | Wt | 2.5 ± 0.6 | 102 ± 8 | 41.1 |
| Ser196 | 2.2 ± 0.4 | 86 ± 4 | 39.1 | |
| Leu196 | 21.5 ± 2.3 | 0.22 ± 0.03 | 0.01 |
a
kcat values were calculated assuming a molecular mass of 170 kDa for the Ffase expressed in S. occidentalis (wild type) and 200 kDa for the enzyme expressed in S. cerevisiae containing Ser or Leu196. The values are given ± standard errors based on the curve fitting using SigmaPlot.