Table 1.
Mutagenesis experiments to interrogate binding of the carboxylate end of fatty acid substrates with hALOX12. Steady state kinetics measurements with AA and DHA reveal only relatively small differences in the catalytic efficiency between wt-hALOX12 and the 2 mutants. Major changes in the positional specificity are observed for H596L but not R402L, with AA.
| Substrate | Arachidonic acid | Docosahexaenoic acid | |||
|---|---|---|---|---|---|
| Enzymes | wt-hALOX12 | H596L | R402L | wt-hALOX12 | R402L |
| KM (µM) | 1.9 ± 0.2 | 1.3 ± 0.4 | 0.89 ± 0.09 | 0.59 ± 0.1 | 1.5 ± 0.5 |
| Kcat (s−1) | 28 ± 0.9 | 8.6 ± 0.7 | 5.6 ± 0.1 | 4.5 ± 0.1 | 5.6 ± 0.5 |
| Kcat/KM (µM)−1s−1 | 14 ± 0.9 | 6.3 ± 0.4 | 6.3 ± 0.5 | 7.6 ± 1.5 | 3.8 ± 1 |
| Product profile (12-HETE:15-HETE:11-HETE) | 100:0:0 | 73:22:5 | 100:0:0 | 29:71a | 37:63a |
a
The product distribution ratio of the DHA reaction is 11-HDHA:14-HDHA.