Table 1.
Steady-state kinetic parameters for selected POP and Pluc variants.
| KM (µM) | kcat (sec-1) | Vmax (RLU) | ||||
|---|---|---|---|---|---|---|
| Variant | −Ni(II) | +Ni(II) | −Ni(II) | +Ni(II) | −Ni(II) | +Ni(II) |
| aPOPWT | 335 ± 10 | 345 ± 13 | 172 ± 2 | 152 ± 3 | NA | NA |
| aPOP167 | 326 ± 22 | 399 ± 32 | 136 ± 4 | 131 ± 5 | NA | NA |
| aPOP517 | 196 ± 18 | 197 ± 19 | 154 ± 5 | 110 ± 4 | NA | NA |
| aPOP167/517 | 219 ± 24 | NA | 181 ± 7 | NA | NA | NA |
| bPlucWT | 1.48 ± 0.15 | 1.19 ± 0.15 | NA | NA | 40200 ± 900 | 37900 ± 1000 |
| bPluc202/532 | 21.5 ± 1.25 | 2.32 ± 0.44 | NA | NA | 20100 ± 400 | 904 ± 38 |
| bPluc108/508 | 0.565 ± 0.145 | 2.19 ± 0.27 | NA | NA | 10600 ± 500 | 1240 ± 40 |
aReactions were conducted in triplicate using 0-1 mM Z-Ala-Pro-pNA and 20-21 nM enzyme in 10% v/v DMSO/30 mM HEPES (pH 7.4) containing 0.8 M NaCl at 85 °C for 1 minute. Average initial rates were determined by changes in absorbance over time at 410 nm using a calculated molar extinction coefficient for pNA (7,126 M-1 cm-1). Kinetic parameters were determined by the non-linear regression function in OriginPro using the Michaelis–Menten equation. bReactions were conducted by mixing 1.1 μM enzyme (preincubated with an equal volume of 1 mM Ni (II) when applicable) with 0–0.75 mM D-luciferin in DMSO (5% v/v DMSO total) in 12.5 mM HEPES (pH 7.8), 5 mM MgSO4, and 1 mM ATP. Luminescence was read for 5 min at room temperature immediately after mixing. The maximum RLU values at each D-luciferin concentration, which describe the “glow” phase of the Pluc reaction mechanism75, were fit to a Michaelis–Menten equation that accounts for inhibition observed at increasing substrate concentrations. This inhibition could be the result of competitive concentrations of inhibitory byproducts58. Max rates describe apparent Vmax of Pluc in the glow phase. Errors for each parameter as calculated in OriginPro from triplicate reactions are provided.