Table 1.
Enzyme | Neu5Ac |
DPAH |
||||
---|---|---|---|---|---|---|
kcat (min− 1) | Km (mM) | kcat/Km (min− 1 mM− 1) | kcat (min− 1) | Km (mM) | kcat/Km (min− 1 mM− 1) | |
Wild type | 260 ± 6a | 4.4 ± 0.3a | 59a | 73 ± 4a | 11 ± 2a | 7a |
E192N | 170 ± 10a | 38 ± 5a | 4.4a | 130 ± 3a | 0.4 ± 0.04a | 340a |
E192Q | 300 ± 20 | 3.5 ± 0.5 | 85 | 170 ± 4 | 0.7 ± 0.05 | 250 |
E192V | 120 ± 8a | 34 ± 5a | 3.5 | 95 ± 4a | 0.4 ± 0.1a | 270a |
E192F | 160 ± 5 | 5.9 ± 0.4 | 27 | 88 ± 2 | 0.3 ± 0.02 | 310 |
E192H | 120 ± 5 | 6.2 ± 0.7 | 19 | 38 ± 1.0 | 0.5 ± 0.04 | 80 |
E192M | 70 ± 4 | 5 ± 0.8 | 14 | 70 ± 3 | 0.2 ± 0.03 | 320 |
E192P | 25 ± 1 | 6 ± 0.9 | 4 | 49 ± 1 | 0.2 ± 0.02 | 260 |
E192D | 120 ± 5 | 4.6 ± 0.5 | 26 | 6 ± 0.1 | 1.1 ± 0.05 | 5 |
E192S | 50 ± 3 | 6 ± 0.8 | 8 | 26 ± 0.7 | 0.6 ± 0.04 | 42 |
The steady-state kinetic parameters of the wild-type and variant enzymes for Neu5Ac and DPAH cleavage were measured with a standard coupled enzyme assay. Kinetic parameters (± standard error of the fit) were determined by fitting the data to the Michaelis–Menten equation.
Data taken from Williams et al.1