TABLE 5.
The pre-steady-state kinetic parameters for substrate hydrolysis by LF apoenzyme activated by divalent metal ions
Pre-steady-state kinetics of substrate cleavage by LF was studied using the stopped-flow method. Prior to reaction initiation, apoenzyme and the mixture containing substrate and divalent metal ions were loaded using two separate syringes to prevent preliminary metal-enzyme and substrate-enzyme interactions. Kinetic parameters were calculated by fitting the experimental data shown in Fig. 4 to Fig. 2D, scheme 4, for Zn2+-activated apoenzyme and Fig. 2E, scheme 5 for Ca2+ and Mn2+ activation. All data were collected in triplicate and fitted using Dynafit software (15). KbindM was calculated as the sum of dissociation constants of each step of metal binding.
| Kinetic constants | Substrate type LF15P |
|||
|---|---|---|---|---|
| Enzyme type LF | Enzyme type apo-LF, metal ion type Zn2+ | Enzyme type apo-LF, metal ion type Ca2+ | Enzyme type apo-LF, metal ion type Mn2+ | |
| k1, m−1 × s−1 | (42.3 ± 6.9) × 106 | (74.6 ± 1.0) × 106 | (88.3 ± 1.1) × 106 | (80 ± 2.0) × 106 |
| k−1, s−1 | 219.3 ± 10.3 | 170.4 ± 2.2 | 270.6 ± 2.7 | 250 ± 5.0 |
| k2, s−1 | 3.9 ± 0.2 | 2.80 ± 0.04 | 2.16 ± 0.03 | 2.0 ± 0.1 |
| k−2, s−1 | 1.3 ± 0.1 | 1.53 ± 0.03 | 1.34 ± 0.02 | 1.1 ± 0.1 |
| k3, s−1 | 0.77 ± 0.04 | 0.71 ± 0.01 | 0.44 ± 0.01 | 0.14 ± 0.01 |
| Km, m | ||||
| Pre-steady state | 1.8 × 10−6 | 1.0 × 10−6 | 1.4 × 10−6 | 1.2 × 10−6 |
| Steady state | (5.4 ± 0.5) × 10−6 | (2.62 ± 0.68) × 10−6 | (3.80 ± 1.28) × 10−6 | (3.67 ± 1.43) × 10−6 |
| kcat, s−1 | ||||
| Pre-steady-state | 0.50 | 0.39 | 0.24 | 0.08 |
| Steady-state | 0.5 ± 0.03 | 0.19 ± 0.02 | 0.18 ± 0.03 | 0.24 ± 0.05 |
| KbindM | 3.9 × 10−6 | 1.2 × 10−4 | 2.2 × 10−5 | |