Table 2. Kinetic parameters for studied mutants.
| Specific activity
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|
| Mutant | Non-l-Phe-activated* | l-Phe-activated† | Activation fold | Km (BH4),‡ μM | Kd (BH4),§ μM | S0.5 (l-Phe),¶ μM | h¶ | Substrate inhibition | Coupling efficiency,∥ % |
| WT | 729 ± 32 | 1,905 ± 12 | 2.6 | 26 ± 3 | 2.7 ± 0.1 | 145 ± 12 | 2.0 | + | 1.03 ± 0.14 |
| F39L | 1,449 ± 24 | 1,703 ± 35 | 1.2 | 44 ± 2 | 8.4 ± 0.8 | 60 ± 8 | 1.4 | + | 1.06 ± 0.08 |
| I65T | 1,600 ± 30 | 2,300 ± 50 | 1.2 | 40 ± 3 | 3.9 ± 0.4 | 80 ± 10 | 1.0 | + | 0.95 ± 0.15 |
| R68S | 1,648 ± 150 | 1,767 ± 134 | 1.1 | 30 ± 3 | 9.0 ± 1.0 | 73 ± 6 | 1.5 | + | 1.18 ± 0.14 |
| H170D | 320 ± 40 | 810 ± 130 | 2.5 | 12 ± 2 | ND | 104 ± 3 | 2.8 | + | ND |
| E178G | 595 ± 119 | 733 ± 74 | 1.2 | 29 ± 5 | ND | 277 ± 38 | 1.1 | + (?) | ND |
| V190A | 500 ± 2 | 2,100 ± 30 | 4.2 | 17 ± 2 | ND | 139 ± 10 | 2.9 | + | ND |
| R261Q | 1,453 ± 56 | 1,485 ± 12 | 1.0 | 25 ± 2 | 2.7 ± 0.1 | 610 ± 60 | 1.1 | - | ND |
| A300S | 500 ± 30 | 590 ± 10 | 1.2 | 26 ± 4 | 2.7 ± 0.1 | 151 ± 25 | 1.1 | + | 0.98 ± 0.22 |
| L308F | 747 ± 9 | 926 ± 28 | 1.2 | 44 ± 8 | ND | 151 ± 13 | 1.9 | + (?) | ND |
| A313T | 650 ± 50 | 1,430 ± 190 | 2.2 | 24 ± 4 | 3.4 ± 0.3 | 165 ± 18 | 1.5 | + | ND |
| A373T | 550 ± 90 | 1,050 ± 110 | 1.9 | 22 ± 3 | ND | 144 ± 14 | 1.8 | + | ND |
| V388M | 280 ± 4 | 440 ± 7 | 1.5 | 24 ± 3 | ND | 1,200 ± 110 | 1.0 | - | ND |
| E390G | 1,370 ± 7 | 1,780 ± 10 | 1.3 | 29 ± 2 | ND | 153 ± 15 | 1.5 | + | ND |
| P407S | 880 ± 60 | 1,800 ± 70 | 2.0 | 17 ± 3 | ND | 140 ± 5 | 2.1 | + | ND |
| Y414C | 653 ± 136 | 1,509 ± 420 | 2.3 | 22 ± 3 | ND | 109 ± 19 | 1.5 | + | ND |
| WT dt-hPAH | - | 1,620 ± 80 | - | 31 ± 1 | ND | 60 ± 5 | 1.0 | + | ND |
| A313T dt-hPAH | - | 1,290 ± 70 | - | 30 ± 3 | ND | 36 ± 3 | 1.2 | + | ND |
Steady-state kinetic parameters of the WT and mutant PAH tetrameric fusion proteins expressed in E. coli. The data include the specific activity with and without prior incubation with l-Phe (activated and non-l-Phe activated), apparent affinity for l-Phe [S0.5 (l-Phe)] and BH4 (Km), and h as a measure of positive cooperativity; equilibrium-binding affinity (Kd) for BH4. ND, not determined.
Obtained with non-l-Phe preincubated enzyme, assayed with 1 mM l-Phe and 75 μM BH4
Obtained with l-Phe preincubated enzyme, assayed with 1 mM l-Phe and 75 μM BH4
Obtained with non-l-Phe preincubated enzyme, assayed with 1 mM l-Phe and variable [BH4] (0-200 μM)
Obtained from equilibrium-binding measurements by isothermal titration calorimetry
Obtained with l-Phe preincubated enzyme, assayed with 75 μM BH4 and variable [l-Phe] (0-4 mM)
Mol l-Tyr produced per mol BH4 oxidized. Data are mean ± SD from five to six independent experiments