TABLE 3.
Kinetic analysis of chondroitinase ABC II site-directed mutants with different substratesThe values, where measurable, are the means of at least three experiments ± standard deviation. NDA, no detectable activity. MDA, minimally detectable activity (activity was too low to allow for reliable determination of kinetic parameters).
|
Enzyme
|
Chondroitin-6-sulfate
|
Dermatan sulfate
|
||||
|---|---|---|---|---|---|---|
| Km | kcat | kcat/Km | Km | kcat | kcat/Km | |
| μm | min–1 | μm–1 min–1 | μm | min–1 | μm–1 min–1 | |
| cABCII | 9.8 ± 2.1 | 1300 ± 113 | 132 | 19.2 ± 2.9 | 1150 ± 26 | 60 |
| H343A | NDA | NDA | NDA | NDA | NDA | NDA |
| H452A | 7.0 ± 1.5 | 10.1 ± 1.9 | 1.4 | 9.1 ± 4.2 | 4.3 ± 0.5 | 0.5 |
| H453A | NDA | NDA | NDA | NDA | NDA | NDA |
| H456A | 10.4 ± 2.4 | 428 ± 51 | 41 | 7.6 ± 1.9 | 144 ± 17 | 19 |
| Y460A | NDA | NDA | NDA | NDA | NDA | NDA |
| R513A | NDA | NDA | NDA | NDA | NDA | NDA |
| E608A | NDA | NDA | NDA | MDA | MDA | MDA |