Table 2.
Metal content and steady-state kinetic constants of Zn(II)-containing L1 analogs.
| Species | Zn(II) content (eq) | kcat(s−1) | Km (µM) |
|---|---|---|---|
| Wild-type L1a | 1.9 ± 0.1 | 27 ± 1 | 3.8 ± 0.5 |
| Wild-type L1 | 1.9 ± 0.1 | 41 ± 1b | 4 ± 1b |
| L1 refolded w/ Zn(II) | 2.0 ± 0.1 | 41 ± 1 | 4.8 ± 0.3 |
| L1 refolded w/o Zn(II) | < 0.03 | < 0.1 | ND |
| L1 refolded w/o Zn(II)c | 2 | 31±1 | 3.6 ± 0.5 |
Steady state kinetic studies were carried out in Chelex-treated 50 mM cacodylate, pH 7.0, using nitrocefin as the substrate.
a
L1 over-expressed in LB medium according to previously published protocol [25].
b
Kinetic reactions conducted in Chelex-treated 50 mM cacodylate, pH 7.0, containing 100 µM Zn(II).
c
Two equivalents of Zn(II) were added to this sample before the steady state kinetic studies were conducted.