Steady-state kinetics of N-acetylation by NAT1 4 and NAT1 11 isozymes expressed in E. coli strain BL21(DE3)pLysS
| Buffer | Component Varied | ApparentKm | Normalized ApparentVmax | Ratio,Vmax/Km | |||
|---|---|---|---|---|---|---|---|
| NAT1 4 | NAT1 11 | NAT1 4 | NAT1 11 | NAT1 4 | NAT1 11 | ||
| μM | nmol of arylamide formed/min/U of NAT1 | ||||||
| Triethanolamine | Substrate | ||||||
| 4-aminosalicylate | 13.6 ± 1.4 | 9.3 ± 0.22-a | 22.2 ± 1.9 | 18.4 ± 0.72-b | 1.6 | 2.0 | |
| Tris | 4-aminosalicylate | 8.9 ± 0.3 | 4.3 ± 0.22-c | 34.6 ± 6.9 | 28.9 ± 4.42-b | 3.9 | 6.7 |
| 5-aminosalicylate | 14.5 ± 1.5 | 7.8 ± 0.52-d | 44.8 ± 3.5 | 39.5 ± 5.42-b | 3.1 | 5.1 | |
| 4-aminobenzoate | 20.8 ± 1.2 | 9.8 ± 0.22-d | 39.2 ± 6.3 | 30.1 ± 7.62-b | 1.9 | 3.1 | |
| Acetyl CoA | 186 ± 8.6 | 258 ± 162-a | 76.7 ± 7.8 | 81.0 ± 242-b | 0.4 | 0.3 | |
Reaction mixtures with cytosol from transformed bacteria (1–2 μg of protein) were prepared in 20 mM Tris-HCl buffer (pH 7.5 at 37°C) containing an acetyl CoA regenerating system and the additional components described under Experimental Procedures. Mixtures with varying amounts of the substrate (2–100 μM) contained a fixed amount of acetyl CoA (0.1 mM); conversely, 4-aminosalicylate was held constant (0.1 mM) when the cofactor was varied from 0.005 to 0.5 mM. Kinetic constants with 4-aminosalicylate as the variable component were also determined with reaction mixtures prepared in 55 mM triethanolamine-HCl buffer (pH 7.5 at 37°C) containing 25 mM KCl and 2.5% (v/v) dimethyl sulfoxide; the acetyl CoA regenerating system and other components were as for the Tris-HCl buffer system. Reactions were at 37°C for 2 to 5 min, depending on the particular substrate or cofactor serving as the variable component, and arylamide products were quantitated by HPLC. NAT1 protein content was ascertained by densitometric scanning of 34-kDa bands on immunoblots, generated with a polyclonal human NAT1 antibody; 1 unit (U) of immunoreactive NAT1 was arbitrarily equated to 1000 relative band density units. ApparentK m and V max constants were calculated with the Cleland Hyper program (Cleland, 1967). The values (mean ± S.D.) represent data from three experiments (three separate bacterial inductions), with triplicate determinations in each experiment. Statistical analyses were performed with the unpaired Student's t test.