Table 2.
Kinetic parameters for the hydrolysis of GalNAc-α-pNP by truncated and full-length BcGH31 and BpGH31, truncated BpGH31 mutants, and BvGH109_1
Values indicate means ± S.E. (n = 3). Reactions were carried out in 50 mm HEPES, pH 7.0, at 37 °C and observed via absorbance at 405 nm. Kinetic parameters were obtained by fitting initial velocities obtained from three replicate assays to the Michaelis–Menten equation using GraFit. Truncated BpGH31 mutants D413A/G/S were inactive.
| Enzyme | kcat | Km | kcat/Km |
|---|---|---|---|
| s−1 | μm | s−1 mm−1 | |
| BcGH31 | 3.6 ± 0.2 | 110 ± 10 | 32 ± 4 |
| tBcGH31 | 2.1 ± 0.1 | 140 ± 20 | 16 ± 2 |
| BpGH31 | 7.2 ± 0.3 | 270 ± 20 | 27 ± 2 |
| tBpGH31 | 2.22 ± 0.09 | 140 ± 20 | 16 ± 2 |
| tBpGH31 D465A | 5.0 ± 0.3 × 10−3 | 42 ± 9 | 0.12 ± 0.02 |
| tBpGH31 D465G | 5.7 ± 0.3 × 10−3 | 340 ± 40 | 0.017 ± 0.002 |
| tBpGH31 D465S | 4.3 ± 0.1 × 10−3 | 46 ± 5 | 0.09 ± 0.01 |
| BvGH109_1 | 0.25 ± 0.01 | 11.5 ± 0.3 | 22.3 ± 0.6 |