TABLE 3.
Comparison of kinetic parameters of Mn2+ and Mg2+ for parental and mutant WecAFLAG-5×Hisa
| Parental or mutant WecAFLAG-His | Mg2+
|
Mn2+
|
||||
|---|---|---|---|---|---|---|
| Vmax (pmol/min/mg) | Km (mM) | Km(N)/Km(E) ratio | Vmax (pmol/min/mg) | Km (mM) | Km(N)/Km(E) ratio | |
| WecAFLAG-His | 23.0 ± 1.3 | 1.7 ± 0.2 | NA | 71.7 ± 2.6 | 0.3 ± 0.04 | NA |
| D90E | 21.3 ± 7 | 9.3 ± 3.8 | 22.3 ± 7.8 | 1.6 ± 0.8 | ||
| D90N | 169.7 ± 43 | 11.0 ± 3.4 | 1.2 | 77.8 ± 14.3 | 0.94 ± 0.3 | 0.6 |
| D91E | 0.6 ± 0.2 | 0.5 ± 0.3 | 0.7 ± 0.1 | 0.08 ± 0.1 | ||
| D91N | 39.4 ± 7 | 5.7 ± 1.4 | 11.4 | 44.3 ± 7 | 0.5 ± 0.1 | 6.2 |
| D159E | 3.1 ± 0.4 | 2.7 ± 0.6 | 2.3 ± 0.3 | 0.6 ± 0.2 | ||
| D159N | 0.9 ± 0.4 | 1.4 ± 1.5 | 0.5 | 2.0 ± 0.4 | 0.3 ± 0.2 | 0.5 |
a
Standard WecAFLAG-His assays were performed with total membrane extracts prepared from E. coli MV501 cells carrying plasmids containing parental or mutant wecA genes. The assays were performed as described in Materials and Methods. Km(N), Km for the protein with an asparagine substitution; Km(E), Km for the protein with a glutamic acid substitution; NA, not applicable.