Table 3.
Kinetic Parameters for the Reaction of rCcP and Its Charge-Reversal Mutants with H2O2.a
| Fast Phase | Slow Phase | Inactiveb | ||||
|---|---|---|---|---|---|---|
| Mutant | % Enzyme | k1 (μM−1s−1) | % Enzyme | k2 (μM−1s−1) | k3 (s−1) | % Enzyme |
| yCcPc | 96 | 45 ± 3 | 0 | - | - | 4 |
| CcP(MI)c | 76 | 47 ± 4 | 12 | - | 11 ± 7 | 12 |
| rCcP | 82 | 48 ± 2 | 3 | 1.3 ± 0.1 | - | 15 |
| Negative Cluster Mutants | ||||||
| R31E | 59 | 44 ± 3 | 0 | - | - | 41 |
| E32K | 98 | 43 ± 6 | 2 | 2.1 ± 0.3 | - | 0 |
| D33K | 94 | 41 ± 7 | 0 | - | - | 6 |
| D34K | 94 | 49 ± 4 | 0 | - | - | 6 |
| E35K | 89 | 44 ± 6 | 3 | 2.2 ± 0.3 | - | 8 |
| D37K | 10 | 37 ± 6 | 2 | - | 6 ± 3 | 88 |
| Mutants Near Ala-193 | ||||||
| E201K | 42 | 43 ± 7 | 0 | - | - | 58 |
| E209K | 27 | 40 ± 12 | 0 | - | - | 73 |
| D210K | 58 | 39 ± 11 | 0 | - | - | 42 |
| Mutants Near Glu-290 | ||||||
| E118K | 57 | 44 ± 8 | 0 | - | - | 43 |
| E290K | 61 | 51 ± 6 | 8 | 2.5 ± 0.4 | - | 31 |
| E291K | 40 | 36 ± 5 | 7 | 1.8 ± 0.3 | - | 53 |
| Top Front-Face Mutants | ||||||
| E17K | 31 | 38 ± 8 | 10 | 2.0 ± 0.3 | - | 59 |
| D18K | 68 | 43 ± 14 | 7 | 2.8 ± 1.5 | - | 25 |
| E98K | 93 | 42 ± 6 | 5 | - | 22 ± 7 | 2 |
a
Experimental conditions: 0.100 M ionic strength, potassium phosphate buffers, pH 7.5, 25°C.
b
Percent inactive enzyme estimated from the increase in the absorbance at 424 nm in the presence of a stoichiometric excess of hydrogen peroxide relative to that for yeast CcP.