Table 4. Steady-State Kinetic Constants for AANATA Site-Directed Mutants.
| Acetyl-CoA | ||||
|---|---|---|---|---|
| varianta,b | Km,app (μM) | kcat,app (s–1) | (kcat/Km)app (M–1 s–1) | (kcat/Km)app-mutant/(kcat/Km)app-wild-type × 100 (%) |
| wild type | 39 ± 12 | 16 ± 1 | (4.1 ± 1.3) × 105 | 100 |
| E47A | 32 ± 3 | 1.26 ± 0.04 | (3.9 ± 0.6) × 104 | 9.5 |
| P48A | 190 ± 10 | 1.4 ± 0.1 | (7.3 ± 0.5) × 103 | 1.8 |
| Y64A | 81 ± 5 | 8.6 ± 0.2 | (1.1 ± 0.1) × 105 | 27 |
| D142A | 80 ± 6 | 27.5 ± 0.6 | (3.4 ± 0.3) × 105 | 83 |
| R153A | 430 ± 60 | 106 ± 6 | (2.5 ± 0.4) × 105 | 61 |
| H178A | 120 ± 7 | 17.9 ± 0.4 | (1.5 ± 0.1) × 105 | 37 |
| C181A | 26 ± 1 | 19.1 ± 0.2 | (7.3 ± 0.3) × 105 | 178 |
| S186A | 29 ± 5 | 5.0 ± 0.3 | (1.7 ± 0.3) × 105 | 41 |
| S182A/S186A | 43 ± 4 | 8.9 ± 0.3 | (2.1 ± 0.2) × 105 | 51 |
| H220A | 160 ± 20 | 4.2 ± 0.2 | (2.6 ± 0.3) × 104 | 6.3 |
| Tyramine | ||||
|---|---|---|---|---|
| varianta,c | Km,app (μM) | kcat,app (s–1) | (kcat/Km)app (M–1 s–1) | (kcat/Km)app-mutant/(kcat/Km)app-wild-type × 100 (%) |
| wild type | 12 ± 1 | 19 ± 1 | (1.6 ± 0.2) × 106 | 100 |
| E47A | 160 ± 50 | 1.0 ± 0.1 | (6 ± 2) × 103 | 0.38 |
| P48A | 470 ± 50 | 0.86 ± 0.04 | (1.84 ± 0.2) × 103 | 0.12 |
| Y64A | 87 ± 17 | 8.8 ± 0.4 | (1.0 ± 0.2) × 105 | 6.3 |
| D142A | 17 ± 1 | 25 ± 1 | (1.5 ± 0.1) × 106 | 94 |
| R153A | 290 ± 20 | 98 ± 2 | (3.4 ± 0.3) × 105 | 21 |
| H178A | 25 ± 3 | 14.7 ± 0.5 | (5.9 ± 0.8) × 105 | 37 |
| C181A | 17 ± 2 | 22 ± 1 | (1.3 ± 0.1) × 106 | 81 |
| S186A | 73 ± 12 | 5.0 ± 0.1 | (6.9 ± 0.6) × 104 | 4.3 |
| S182A/S186A | 190 ± 20 | 8.3 ± 0.2 | (4.3 ± 0.3) × 104 | 2.7 |
| H220A | 82 ± 9 | 4.1 ± 0.2 | (4.9 ± 0.6) × 104 | 3.1 |
a
Kinetic constants are reported with the standard error (n = 3).
b
The reaction rate was measured at a fixed saturating concentration of tyramine while varying the concentration of acetyl-CoA.
c
The reaction rate was measured at a fixed saturating concentration of acetyl-CoA while varying the concentration of tyramine.