Table 3.
Enzymatic activity of Lon fragments containing catalytic sites and of full-length enzyme
| Enzyme or fragment | ATPasea | Thioesterase | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| (-) β-casein | (+) β-casein | |||||||||
| I | II | II | (−) ATP/Mg | (+) ATP/Mg | ||||||
| kcat’ rnin−1 | km’ rnM | kcat’ mm−1 | km’mM | kcat’ mm−1 | km’ mM | kcat’ s−1 | km’ mM | kcat’ s−1 | km’ mM | |
| Lon-w.t. | 110 ± 8 | 0.60 ± 0.05 | 40 ± 5 | 0.20 ± 0.04 | 120 ± 10 | 0.20 ± 0.05 | 4.2 ± 0.4 | 3.5 ± 0.01 | 50 ± 4 | 3.0 ± 0.02 |
| Lon-S679A | 100 ± 5 | 0.50 ± 0.07 | 33 ± 3 | 0.15 ± 0.05 | 100 ± 8 | 0.20 ± 0.04 | inactive | |||
| A | inactive | |||||||||
| AP-w.t. | 33 ± 2 | 0.30 ± 0.06 | 35 ± 2 | 0.30 ± 0.04 | 30 ± 2 | 0.25 ± 0.06 | 0.7 ± 0.1 | 0.4 ± 0.07 | 13 ± 1 | 0.4 ± 0.06 |
| AP-S679A | 25 ± 3 | 0.25 ± 0.08 | 30 ± 4 | 0.20 ± 0.05 | 28 ± 2 | 0.30 ± 0.06 | inactive | |||
| P-w.t. | 0.04 + 0.01 | 0.30 + 0.08 | ||||||||
a
ATPase activity determined at the ATP-Mg ratio 1:1 (condition I) or in the presence of constant excess of magnesium ions (15 mM concentration, condition II). Kinetic parameters were derived from linear double-reciprocal plots 1/V-1/[S]. The values of the apparent catalytic constant kcat were calculated using concentration of protein (as monomer) estimated by the predicted molar absorbance coefficient (http://au.expasy.org/tools/protparam.html) and the absorbance at 280 nm.