Table 4. The activity of PLP form of TaTT variants in half-reaction with R-PEA.
| TaTT | Mutations | pH 8.0, Tris-HCl buffer, 30°C | pH 9.0, CHES buffer, 40°C | ||||
|---|---|---|---|---|---|---|---|
| kmax, | KD, | kmax/KD, | kmax, | KD, | kmax/KD, | ||
| s-1 | mM | s-1M-1 | s-1 | mM | s-1M-1 | ||
| WT | No | 0.030 ± 0.001 | 82 ± 6 | 0.36 ± 0.03 | 0.073 ± 0.004 | 81 ± 8 | 0.9 ± 0.1 |
| mP1 | R43S | 0.030 ± 0.002 | 110 ± 13 | 0.27 ± 0.04 | n.m. | n.m. | n.m. |
| mP2 | G41V + Y101F | No saturation | 2.1 ± 0.5 | 0.28 ± 0.02 | 13 ± 2 | 20 ± 3 | |
| mP3 | R43S+ G41V + Y101F | No saturation | 10.0 ± 0.5 | 0.15 ± 0.01 | 2.6 ± 0.4 | 60 ± 10 | |
| mO1 | S115R | 0.050 ± 0.004 | 48 ± 8 | 1.0 ± 0.2 | 0.011 ± 0.001 | 3.3 ± 1.2 | 3.3 ± 1.2 |
| mO2 | Y166W + F39Y | n.d. | n.d. | ||||
| mP2O1 | G41V + Y101F + S115R | No saturation | 5.7 ± 0.2 | n.m. | n.m. | n.m. | |
| mP3O1 | R43S + G41V + Y101F + S115R | No saturation | 35 ± 1 | 0.13 ± 0.01 | 1.5 ± 0.5 | 90 ± 30 | |
| mP3O3 | R43S + G41V + Y101F + S115R + A108 | 0.9 ± 0.2 | 101 ± 22 | 9 ± 3 | n.m. | n.m. | n.m. |
| mP3O4 | R43S + G41V + Y101F + Y166W + F39Y | n.d. | n.d. | ||||
| mP3O5 | R43S + G41V + Y101F + W32H | No saturation | 6.5 ± 0.8 | n.m. | n.m. | n.m. | |
| mP3O6 | R43S + G41V + Y101F + F39Y | n.d. | n.d. | ||||
| mP3O7 | R43S + G41V + Y101F + F39Y + W32H | 0.011 ± 0.001 | 44 ± 8 | 0.25 ± 0.05 | n.m. | n.m. | n.m. |
Variants containing changes in P-pocket are named mP1-mP3, variants containing changes in O-pocket are named mO1-mO4; variants with changes in both pockets are named mPxOx accordingly.
n.m. = not measured; n.d. = not detected