Table 2.
Steady-state kinetic constants for wild-type and mutant BT2127-catalyzed hydrolysis of pyrophosphate at 25 °C and pH 7.5. See Materials and Methods for Details.
| BT2127 | kcat (min−1) | Km (μM) | kcat/Km (M−1s−1) |
|---|---|---|---|
| Wild-type | 1.9 (± 0.06) × 101 | 3.6 ± 0.4 | 8.9 × 104 |
| D11N | NAa | ------ | ------ |
| D13A | NAa | ------- | ------ |
| D13N | NAa | ------- | ------ |
| K147A | ∼2 × 10−3b | ------- | ------ |
| N172A | 7.4 (± 1.8) × 10−1 | 4.5 ± 0.3 | 3 × 103 |
| T113A | 4.0 (± 0.03) × 10−1 | 5.9 ± 0.2 | 1 × 103 |
| S115A | 8.4 (± 0.12) ×10−2 | 3.6 ± 0.4 | 4 × 102 |
| T50A | 4.1 (± 0.18) × 10−1 | 2.7 ± 0.7 | 3 × 103 |
| R49A | ∼3 × 10−2b | ------- | ------- |
| K79A | ∼2 × 10−2b | ------- | ------- |
| H23A | 3.3 (± 0.06) × 10−1 | 1.2 (± 0.08) × 101 | 5 × 102 |
| M20A | ∼5.1 × 10−2b | ------- | ------- |
| M20K | 2.4 (± 0.04) × 10−1 | 2.5 (±0.3) × 101 | 2 × 102 |
| M20L | 5.8 (± 0.04) × 10−2 | 3.5 ± 0.1 | 3 × 102 |
| E47A | ∼3 × 10−3 | ------- | ------- |
| E47Q | 4.8 (± 0.4) × 10−1 | 6 ± 1 | 1 × 103 |
| E47N | ∼3 × 10−3b | ------- | ------- |
| E47D | ∼3 × 10−3b | ------- | ------- |
| S80A | 1.7 (± 0.04) × 10−1 | 7.1 ± 0.9 | 4.1 × 102 |
| Q117A | ∼3 × 10−2b | ------- | ------- |
| Y76F | ∼2 × 10−2b | ------- | ------- |
| S19A | ∼2 × 1 0−2b | ------- | ------- |
a
NA represents no activity detected above the detection limit kcat < 0.001 min−1.
b
The rate was determined by measuring the initial velocity of orthophosphate formation (μM/min) and dividing that value by the enzyme concentration. Reaction solutions initially contained 10 μM enzyme, 300 μM pyrophosphate, 1 mM MgCl2, 1.0 unit/ml purine nucleoside phosphorylase, and 0.2 mM MESG in 50 mM Tris (pH 7.5).