TABLE 1.
Steady-state kinetic parameters for the direct assay of hydrolyase activity
Reactions containing substrate and buffer salts were preincubated at 60 °C before the addition of reconstituted MJ1003/MJ1271 protein to initiate the reaction. Hydrolase activity was determined by measuring the rate of decrease in UV absorbance caused by the unsaturated substrate, as described under “Experimental Procedures.”
| Substrate | Km | Vmax | kcat | kcat/Km |
|---|---|---|---|---|
| μm | units mg-1 | s-1 | m-1 s-1 | |
| cis-Homo1aconitate | 22 ± 3 | 0.68 ± 0.03 | 0.75 | 3.4 × 104 |
| cis-Homo2aconitate | 30 ± 5 | 0.60 ± 0.03 | 0.66 | 2.2 × 104 |
| cis-Homo3aconitate | 36 ± 6 | 2.2 ± 0.09 | 2.5 | 6.8 × 104 |
| cis-Homo4aconitate | 175 ± 39 | 5.1 ± 0.3 | 5.6 | 3.2 × 104 |
| Maleate | 330 ± 50 | 5.5 ± 0.3 | 6.0 | 1.8 × 104 |
| (R)-Homocitratea | 1500 ± 200 | 0.59 ± 0.03 | 0.37 | 2.5 × 102 |
a
Dehydratase activity was determined by measuring the rate of increase in UV absorbance caused by homoaconitate formation.