FIG. 5.
Steady-state kinetics of the LuxG reaction. A primary double-reciprocal plot of initial rates of the His6-tagged LuxG reactions versus various concentrations of NADH and FMN. Each line represents e/V values at a fixed concentration of FMN. Assay reactions were performed in 50 mM Tris-Cl (pH 8), 10% (vol/vol) glycerol, 1 mM DTT, and 100 nM concentrations of His6-tagged LuxG with various concentrations of FMN as indicated (1 to 40 μM) and NADH (5 to 200 μM) using a stopped-flow apparatus at 4°C under aerobic conditions. The Dalziel parameters (7, 9) were determined: φ[NADH] = 8.97 μM s, φ[FMN] = 1.60 μM s, and φo = 0.59 s.