TABLE 2.
Comparison of kinetic parameters of UDP-GlcNAc for parental and mutant WecAFLAG-5×His
| Parental or mutant WecAFLAG-5×His plasmid (protein form) | Vmax (pmol/min/mg)a | Km (μM) | Relative Vmax/Km |
|---|---|---|---|
| pKV1 | 56.4 ± 3.5 | 0.19 ± 0.05 | 100 (297)b |
| pKV2 (D90E) | 23.6 ± 2.3 | 0.18 ± 0.07 | 43 (129) |
| pKV3 (D90N) | 15.5 ± 2.0 | 0.12 ± 0.09 | 44 (130) |
| pKV4 (D91E) | 32.7 ± 2.0 | 0.03 ± 0.02 | 392 (1,167) |
| pKV5 (D91N) | 42.4 ± 3.8 | 0.17 ± 0.06 | 81 (242) |
| pKV8 (D156E) | ND | ND | NA |
| pKV9 (D156N) | ND | ND | NA |
| pKV10 (D159E) | 5.9 ± 3.1 | 1.97 ± 1.55 | 1.0 (3) |
| pKV11 (D159N) | 6.0 ± 3.3 | 1.84 ± 1.53 | 1.0 (3) |
a
Standard WecAFLAG-His assays were performed with total membrane extracts prepared from MV501 E. coli cells carrying the plasmids containing parental or mutant wecA genes with 10 times the concentration corresponding to the experimentally determined Km for Mn2+, as described in Materials and Methods. ND, no detectable activity above the background; NA, not applicable.
b
The numbers in parentheses are the absolute values of dividing Vmax by Km.