Steady-state kinetics of hAOX1 and its variants corresponding to SNPs
Benzaldehyde, phthalazine, phenanthridine, and chloroquinazolinone were used as substrates to cover a range from aldehydes to N-heterocyclic compounds. Assays were performed photometrically using 2,6-dichlorphenolindophenol as a final electron acceptor. With phenanthridine, molecular oxygen was used as the electron acceptor. Data are mean values from three independent measurements (±S.D.).
| Substrate | Protein Portion | Kinetic Parameters | WT | R802C | R921H | N1135S | H1297R |
|---|---|---|---|---|---|---|---|
| Benzaldehyde | KM, μM | 7.1 ± 0.6 | 7.6 ± 1.9 | 6.3 ± 1.2 | 6.7 ± 2.8 | 5.2 ± 1.8 | |
| Active portion | kcat, min−1 | 6.4 ± 0.1 | 5.3 ± 0.3 | 1.7 ± 0.1 | 6.2 ± 0.3 | 6.4 ± 0.3 | |
| kcat/KM, 1/min · μM | 0.91 ± 0.17 | 0.70 ± 0.16 | 0.27 ± 0.08 | 0.92 ± 0.11 | 1.23 ± 0.17 | ||
| Total protein | kcat, min−1 | 2.7 ± 0.1 | 1.8 ± 0.3 | 1.1 ± 0.1 | 3.8 ± 0.3 | 2.6 ± 0.3 | |
| kcat/KM, 1/min · μM | 0.38 ± 0.17 | 0.24 ± 0.16 | 0.17 ± 0.08 | 0.57 ± 0.11 | 0.50 ± 0.17 | ||
| Phthalazine | KM, μM | 1.3 ± 0.3 | 0.9 ± 0.3 | 1.6 ± 0.3 | 1.2 ± 0.1 | 1.3 ± 0.2 | |
| Active portion | kcat, min−1 | 5.6 ± 0.2 | 5.2 ± 0.3 | 2.4 ± 0.1 | 7.2 ± 0.1 | 5.4 ± 0.1 | |
| kcat/KM, 1/min · μM | 4.31 ± 0.67 | 5.78 ± 1.00 | 1.50 ± 0.33 | 6.00 ± 1.00 | 4.15 ± 0.50 | ||
| Total protein | kcat, min−1 | 2.3 ± 0.2 | 1.8 ± 0.3 | 1.5 ± 0.1 | 4.4 ± 0.1 | 2.9 ± 0.1 | |
| kcat/KM, 1/min · μM | 1.77 ± 0.67 | 2.00 ± 1.00 | 0.94 ± 0.33 | 3.67 ± 1.00 | 2.23 ± 0.50 | ||
| Phenanthridine | KM, μM | 3.9 ± 0.8 | 4.4 ± 0.4 | N.D. | 6.1 ± 1.0 | 4.1 ± 0.7 | |
| Active portion | kcat, min−1 | 12.2 ± 0.5 | 10.2 ± 0.2 | N.D. | 32.6 ± 1.1 | 31.5 ± 1.0 | |
| kcat/KM, 1/min · μM | 3.13 ± 0.63 | 2.32 ± 0.50 | 5.34 ± 1.10 | 7.68 ± 1.43 | |||
| Total protein | kcat, min−1 | 5.3 ± 0.5 | 3.4 ± 0.2 | N.D. | 19.9 ± 1.1 | 16.9 ± 1.0 | |
| kcat/KM, 1/min · μM | 1.36 ± 0.63 | 0.77 ± 0.50 | 3.26 ± 1.10 | 4.12 ± 1.43 | |||
| Chloroquinazolinone | KM, μM | 5.2 ± 0.7 | 4.7 ± 0.7 | 4.5 ± 0.8 | 4.1 ± 0.5 | 5.8 ± 0.5 | |
| Active portion | kcat, min−1 | 5.6 ± 0.1 | 5.4 ± 0.2 | 3.6 ± 0.1 | 6.5 ± 0.1 | 6.7 ± 0.2 | |
| kcat/KM, 1/min · μM | 1.08 ± 0.14 | 1.15 ± 0.29 | 0.80 ± 0.13 | 1.59 ± 0.20 | 1.16 ± 0.4 | ||
| Total protein | kcat, min−1 | 2.3 ± 0.1 | 1.8 ± 0.2 | 2.2 ± 0.1 | 4.0 ± 0.1 | 3.6 ± 0.2 | |
| kcat/KM, 1/min · μM | 0.44 ± 0.14 | 0.38 ± 0.29 | 0.49 ± 0.13 | 0.98 ± 0.20 | 0.62 ± 0.4 | ||
| Oligomerization in solution | Dimer | Percentage | 58 | 39 | 83 | 90 | 78 |
| Monomer | Percentage | 38 | 58 | 13 | 7 | 17 | |
| Multimer | Percentage | 4 | 3 | 4 | 3 | 5 |
N.D., no activity was detectable; WT, wild type.