Table 3.
Regiospecificity of Wild-type and Mutant hHO1 in the Reaction Supported by Sodium Ascorbate in the Absence or Presence of SOD and Catalase.a
| enzyme | biliverdin isomers (%) | biliverdin isomers (%) | ||||
|---|---|---|---|---|---|---|
| hHO1 + sodium ascorbate | hHO1 + sodium ascorbate + SOD + catalase | |||||
| α | β | δ | α | β | δ | |
| wild-type | 100 | – | – | 100 | – | – |
| D140A | 94.0 ± 1.5 | 2.7 ± 0.6 | 3.3 ± 1.5 | 94.7 ± 2.3 | 2.0 ± 0.1 | 3.3 ± 1.7 |
| D140H | 83.0 ± 10.5 | 7.0 ± 4.2 | 10.0 ± 6.0 | 90.4 ± 4.4 | 4.1 ± 1.5 | 5.5 ± 4.2 |
| D140K | 91.7 ± 1.5 | 3.3 ± 2.1 | 5.0 ± 2.6 | 96.5 ± 0.6 | 1.3 ± 0.6 | 2.2 ± 1.0 |
| Y58F | 99.3 ± 0.6 | 0.3 ± 0.5 | 0.4 ± 0.3 | N.D.d | N.D. | N.D. |
| Y58A | 100 | – | – | N.D. | N.D. | N.D. |
| Y58A/D140A | 96.7 ± 1.5 | 1.3 ± 0.6 | 2.0 ± 0.6 | N.D. | N.D. | N.D. |
| K18A | 99.5 ± 0.6 | – | 0.5 ± 0.5 | N.D. | N.D. | N.D. |
| K18E | 91.6 ± 4.7 | 1.2 ± 0.2 | 7.2 ± 4.7 | 97.5 ± 0.2 | 1.5 ± 0.2 | 1.5 ± 0.1 |
| E29Kb | 83.8 ± 0.8 | 6.7 ± 0.4 | 8.4 ± 0.3 | 95.6 ± 0.4 | 2.3 ± 0.3 | 2.2 ± 0.2 |
| R183A | 96.7 ± 2.5 | – | 3.3 ± 2.5 | N.D. | N.D. | N.D. |
| R183Ec | 80.0 ± 3.6 | 0.7 ± 0.5 | 19.3 ± 3.1 | 91.2 ± 1.0 | 0.8 ± 1.0 | 8.0 ± 0.8 |
| Y134F | 99.7 ± 0.6 | – | 0.4 ± 0.5 | N.D. | N.D. | N.D. |
| Y134F/R183E | 73.3 ± 6.4 | 1.7 ± 1.2 | 25.0 ± 5.3 | 76.7 ± 3.8 | 4.0 ± 2.0 | 19.3 ± 2.9 |
| K18A/R183E | 51.0 ± 1.7 | 2.3 ± 0.6 | 46.7 ± 1.5 | 55.7 ± 0.6 | 2.0 ± 0.1 | 42.3 ± 0.6 |
| K18E/R183E | 20.5 ± 3.7 | 2.6 ± 0.2 | 76.9 ± 3.6 | 21.1 ± 1.8 | 2.5 ± 0.0 | 76.4 ± 1.8 |
| K18A/Y134F/R183E | 49.0 ± 4.5 | 3.0 ± 1.0 | 48.0 ± 5.1 | 52.7 ± 1.5 | 3.0 ± 0.1 | 44.3 ± 1.5 |
| K18E/E29K/R183E | 8.0 ± 2.7 | 12.9 ± 3.9 | 79.1 ± 5.7 | 10.2 ± 0.5 | 3.4 ± 0.3 | 86.4 ± 0.5 |
a
All experiments were repeated at least three times.
b
E29K mutant also generated 1.1 ± 0.1% of biliverdin IXγ, which was not observed for the other mutants.
c
Reference (42).
d
N.D. = not determined.