Table 1 Steady-state kinetic parameters for selected POP and Pluc variants.

^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 V_max of Pluc in the glow phase. Errors for each parameter as calculated in OriginPro from triplicate reactions are provided.

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