TABLE 3.
Apparent kinetic parameters of recombinant LuxG and His6-tagged LuxG from P. leiognathi TH1
| Substratea | Second substrate (concn [μM]) | Mean ± SDb
|
|
|---|---|---|---|
| Apparent Km (μM) | kcat (s−1) | ||
| FMN* | NADH (200) | 2.7 ± 0.3 (3.2 ± 0.3) | 10.6 ± 0.3 (11.3 ± 0.3) |
| FAD* | NADH (200) | 2.6 ± 0.4 (3.4 ± 0.4) | 7.1 ± 0.3 (8.0 ± 0.2) |
| Riboflavin* | NADH (200) | 1.2 ± 0.1 (1.2 ± 0.2) | 9.6 ± 0.2 (8.0 ± 0.3) |
| NADH† | FMN (40) | 20.5 ± 2.9 (19.6 ± 2.2) | 7.5 ± 0.3 (7.6 ± 0.2) |
a
Initial rates were measured by the flavin reductase assay described in Materials and Methods at 25°C and pH 8 with 0.2 μM concentrations of recombinant LuxG or His6-tagged LuxG, flavins (2 to 40 μM) at a fixed concentration of NADH (200 μM) or various concentrations of NADH (5 to 200 μM) at a fixed concentration of FMN (40 μM). Experiments were performed by using a stopped-flow spectrophotometer (*) or a standard spectrophotometer (†). Data were analyzed by using nonlinear regression analysis (Kaleidagraph software; Synergy).
b
The kinetic parameters of His6-tagged LuxG are shown in parentheses.