TABLE 2.
Steady state kinetic parameters of ALR, CBL, and their evolved variants
All values are the mean ± S.E. for three independent experiments (each including duplicate assays), except for the ALR(Y274F) assays with cystathionine, in which n = 6. The kcat values are reported per active site.
| Enzyme | Cystathionine → homocysteine + Pyruvate + NH3 |
l-Ala → d-Ala |
||||
|---|---|---|---|---|---|---|
| kcat | Km | kcat/Km | kcat | Km | kcat/Km | |
| s−1 | mm | s−1·m−1 | s−1 | mm | s−1·m−1 | |
| ALR | NDa | NDa | <0.85 | 55 ± 4 | 0.87 ± 0.15 | 6.4 × 104 |
| ALR(Y274F) | (2.8 ± 0.3) × 10−3 | 3.3 ± 1.0 | 0.85 | 40 ± 5 | 1.5 ± 0.6 | 2.6 × 104 |
| CBL | 90 ± 14 | (3.9 ± 0.7) × 10−2 | 2.3 × 106 | 3.3 ± 0.6 | 51 ± 4 | 65 |
| CBL(P113S) | 95 ± 3 | 0.12 ± 0.02 | 7.9 × 105 | 21.6 ± 0.4 | 58 ± 3 | 3.7 × 102 |
| CBL(P113A) | 81 ± 11 | 0.31 ± 0.07 | 2.6 × 105 | 4.6 ± 0.5 | 23 ± 5 | 2.0 × 102 |
| CBL(P113T) | 30 ± 2 | 0.21 ± 0.02 | 1.4 × 105 | 5.8 ± 0.5 | 39 ± 4 | 1.5 × 102 |
| CBL(P113Q) | 96 ± 8 | 0.12 ± 0.02 | 8.0 × 105 | 3.6 ± 0.6 | 33 ± 10 | 1.1 × 102 |
| CBL(Y111F) | 0.07 ± 0.01 | (2.3 ± 1.9) × 10−2 | 3.1 × 103 | NDa | NDa | ∼0.011 |
a ND means, not detected.