TABLE 2.
Kinetic parameters of Ape1a and mutant variants
Reactions were conducted in triplicate in 50 mm sodium phosphate buffer, pH 6.5, at 25 °C using p-nitrophenyl acetate as substrate over a concentration range of 0.1–6 mm.
| Enzyme | Km | kcat | kcat/Km |
|---|---|---|---|
| mm | s−1 | mol−1·s−1 | |
| Wild-type | 0.60 ± 0.099 | 8.7 ± 0.40 | 14,500 ± 2480 (100%)a |
| Catalytic triad | |||
| S80A | 2.2 ± 0.57 | 0.0084 ± 0.0013 | 3.8 ± 1.14 (0.026%) |
| D366A | 0.74 ± 0.087 | 0.21 ± 0.0082 | 280 ± 34 (1.9%) |
| H369A | 1.1 ± 0.077 | 0.0078 ± 0.00019 | 7.4 ± 0.59 (0.051%) |
| Potential binding residues | |||
| G78A | 0.72 ± 0.20 | 0.43 ± 0.032 | 590 ± 160 (4.1%) |
| D79A | 0.39 ± 0.12 | 0.042 ± 0.0035 | 110 ± 24 (0.73%) |
| H81F | 0.68 ± 0.26 | 3.4 ± 0.88 | 4900 ± 1500 (34%) |
| N235A | 0.96 ± 0.27 | 1.9 ± 0.17 | 2000 ± 390 (13%) |
| G236A | 0.69 ± 0.089 | 2.5 ± 0.093 | 3600 ± 470 (25%) |
| T267A | 0.58 ± 0.094 | 0.83 ± 0.038 | 1400 ± 230 (9.8%) |
| N268L | 0.84 ± 0.12 | 0.026 ± 0.0010 | 30 ± 4.4 (0.21%) |
| N268Q | 0.42 ± 0.084 | 0.087 ± 0.0044 | 220 ± 35 (1.4%) |
| N268S | 1.7 ± 0.26 | 0.12 ± 0.0068 | 67 ± 10 (0.46%) |
| V368A | 0.91 ± 0.29 | 1.8 ± 0.18 | 2000 ± 460 (14%) |
a Percentages in parentheses were calculated relative to wild-type Ape1a.