Table 1. Kinetic Parameters of Wild-Type OvoATh2 and Its Mutants under Different Conditions.
| enzyme | substrates | kcat, O2 [min–1] | KM, l-His or l-His derivatives [μM] | KM, l-Cys [μM] | kcat/KM, l-His or l-His derivatives or l-Cys [min–1μM–1] | % of coupling product from NMR analysis |
|---|---|---|---|---|---|---|
| wild-type | l-His + l-Cys | 589.0 ± 3.5 | 585.1 ± 28.0 | 278.6 ± 9.0 | 2.10 ± 0.09 | ∼90% |
| wild-type | l-Her + l-Cys | 127.2 ± 1.5 | 61.9 ± 3.5 | (1.86 ± 0.04) E3 | 66.9 ± 2.3 | ∼50% |
| wild-type | l-Cys | 26.7 ± 0.4 | (2.6 ± 0.2) E3 | below the detection limit | ||
| wild-type | 3-Me-l-His + l-Cys | 131.2 ± 2.7 | 1.9 ± 0.1 | 252.2 ± 14.5 | 0.52 ± 0.04 | below the detection limit |
| wild-type | 1-Me-l-His + l-Cys | 20.5 ± 0.3 | (3.9 ± 0.1) E3 | (5.3 ± 0.1) E-3 | below the detection limit |
| enzyme | substrates | kcat, O2 [min–1] | KM, l-His [μM] | KM, l-Cys [μM] | kcat/KM, l-Cys [min–1 μM–1] | % of coupling product from NMR analysis |
|---|---|---|---|---|---|---|
| Y406F | l-His + l-Cys | 361.2 ± 5.9 | 38.2 ± 2.6 | 575.2 ± 22.0 | 0.63 ± 0.09 | ∼20% |
| Y68F | l-His + l-Cys | 23.4 ± 0.2 | 50.9 ± 2.5 | (61.9 ± 1.5) E3 | (0.4 ± 0.1) E-3 | ∼30% |
| Y68F/Y406F | l-His + l-Cys | 14.8 ± 0.3 | 31.3 ± 4.0 | (125.7 ± 11.0) E3 | (1.2 ± 0.1) E-4 | ∼10% |