Table I.
Biochemical Properties of BluB Point Mutants
| BluB mutant | DMB produced (nmol)ab | Kd for FMN (μM)a | ΔGb for FMNox (kcal mol−1)ac | Kd for FMNH2 (μM)a | ΔGb for FMNH2 (kcal mol−1)ac |
|---|---|---|---|---|---|
| Wild type | 8.3 ± 0.1 | 6.9 ± 0.3 | −7.2 ± 0.02 | 0.62 ± 0.12 | −8.6 ± 0.1 |
| Lid domain, active site closure | |||||
| M94I | 1.5 ± 0.1 | 29 ± 3 | −6.3 ± 0.1 | 1.6 ± 0.2 | −8.0 ± 0.1 |
| G110S | – | 38 ± 3 | −6.1 ± 0.04 | 4.5 ± 0.9 | −7.4 ± 0.1 |
| Near FMN phosphate group | |||||
| R30H | – | 31 ± 4 | −6.2 ± 0.1 | 82 ± 21d | −5.7 ± 0.2 |
| A57V | – | 24 ± 3 | −6.4 ± 0.1 | 170 ± 44d | −5.2 ± 0.2 |
| P58L | – | 24 ± 3 | −6.4 ± 0.1 | 1.7 ± 0.1 | −8.0 ± 0.04 |
| P202L | – | 28 ± 4 | −6.3 ± 0.1 | 4.1 ± 0.9 | −7.5 ± 0.1 |
| Conserved loop near active site | |||||
| G61D | – | 44 ± 5 | −6.0 ± 0.1 | 5.3 ± 0.8d | −7.3 ± 0.1 |
| Near N5 of FMN | |||||
| G133S | – | 32 ± 2 | −6.2 ± 0.04 | 4.3 ± 0.8 | −7.4 ± 0.1 |
| M140I | 0.68 ± 0.06 | 27 ± 3 | −6.3 ± 0.1 | 4.3 ± 1.1 | −7.4 ± 0.1 |
| S167G | 0.60 ± 0.07 | 34 ± 2 | −6.2 ± 0.04 | 2.1 ± 0.2 | −7.9 ± 0.1 |
| Near N1 of FMN | |||||
| D32N | 0.14 ± 0.02 | < 0.10e | < −9.7e | < 0.10e | < −9.7e |
| Double mutant | |||||
| D32N/S167G | – | < 0.10e | < −9.7e | < 0.10e | < −9.7e |
a
Error represents standard error.
b
Results of three independent experiments. –, none detected; detection limit is 0.12 nmol.
c
ΔGb, free energy of binding, calculated from Kd.
d
Data were weighted in KaleidaGraph to achieve reasonable curve fits.
e
Binding curves indicate tight binding; exact Kd and ΔGb values cannot be calculated.