TABLE 3.
Steady-state kinetic parameters for Xyl3B wild-type and mutant enzymes
| Enzyme | Mean value ± SEMa |
|||||
|---|---|---|---|---|---|---|
|
pNP-β-d-Xylopyranoside |
pNP-β-d-Glucopyranoside |
|||||
| kcat (s−1) | Km (mM) | kcat/Km (s−1 mM−1) | kcat (s−1) | Km (mM) | kcat/Km (s−1 mM−1) | |
| Xyl3B WT | 250 ± 10 | 8.4 ± 1 | 30 ± 4 | 19 ± 0.6 | 22 ± 1 | 0.86 ± 0.05 |
| Xyl3B E115A | 0.35 ± 0.02 | 28 ± 3 | (1.3 ± 0.2) × 10−2 | (8.7 ± 0.5) × 10−2 | 39 ± 4 | (2.2 ± 0.3) × 10−3 |
| Xyl3B E115D | 87 ± 5 | 39 ± 4 | 2.2 ± 0.2 | 16 ± 0.4 | 17 ± 1 | 0.94 ± 0.06 |
| Xyl3B R177Ab | 0.17 ± 0.01 | 8.8 ± 1 | (1.9 ± 0.2) × 10−2 | ND | ND | ND |
| Xyl3B K214Ab | 0.10 ± 0.01 | 5.7 ± 0.9 | (1.8 ± 0.3) × 10−2 | ND | ND | ND |
| Xyl3B H215A | 4.0 ± 0.2 | 19 ± 1 | 0.21 ± 0.02 | (3.8 ± 0.4) × 10−2 | 23 ± 4 | (1.7 ± 0.3) × 10−3 |
| Xyl3B M251Ab | 75 ± 6 | 100 ± 10 | 0.75 ± 0.1 | ND | ND | ND |
| Xyl3B D286Ab,c | (1.20 ± 0.3) × 10−2 | ND | ND | ND | ND | ND |
a
The data are reported as means ± standard errors from the mean for three independent experiments.
b
Kinetic parameters for pNPG were not determined because the activity was below the limit of sensitivity for the assay.
c
The reported kcat value is kcat(apparent), as Km values were not determined; rates for this mutant were determined at a 46.25 mM pNPX concentration and thus represent a lower boundary on kcat.