TABLE 4.
Kinetic parameters of wild-type and Gly357 mutant human APAs toward various aminoacyl-MCA
Wild-type or G357H APAs (50 ng/ml) were incubated with aminoacyl-MCAs at 37 °C for 5 min with or without 1 mm CaCl2. Kinetic parameters were determined from Lineweaver-Burk plots.
| Substrate | APA | CaCl2 | Kma | kcata | kcat/Km × 103s |
|---|---|---|---|---|---|
| 1 mm | μm | s−1 | μm−1·s−1 | ||
| Glu-MCA | Wild type | − | 808 ± 68 | 6.89 ± 0.38 | 8.53 ± 0.28 |
| G357H | − | 148 ± 7.6 | 11.6 ± 0.75 | 78.0 ± 6.0 | |
| Asp-MCA | Wild type | − | 3040 ± 290 | 5.61 ± 0.33 | 1.85 ± 0.080 |
| G357H | − | 933 ± 90 | 9.49 ± 1.1 | 10.2 ± 0.28 | |
| Gln-MCA | Wild type | − | 852 ± 67 | 4.25 ± 0.23 | 4.98 ± 0.21 |
| G357H | − | 338 ± 18 | 11.0 ± 0.39 | 32.7 ± 0.72 | |
| Ala-MCA | Wild type | − | 234 ± 31 | 1.64 ± 0.18 | 7.00 ± 0.39 |
| G357H | − | 461 ± 27 | 7.37 ± 0.30 | 16.0 ± 1.2 | |
| Glu-MCA | Wild type | + | 201 ± 23 | 18.7 ± 0.29 | 90.3 ± 1.3 |
| G357H | + | 90.5 ± 6.6 | 34.2 ± 2.7 | 378 ± 22 | |
| Asp-MCA | Wild type | + | 1275 ± 69 | 10.8 ± 0.72 | 8.45 ± 0.63 |
| G357H | + | 292 ± 22 | 16.5 ± 0.56 | 56.5 ± 5.7 | |
| Gln-MCA | Wild type | + | 1288 ± 69 | 4.00 ± 0.22 | 3.10 ± 0.058 |
| G357H | + | 457 ± 55 | 5.17 ± 0.35 | 11.3 ± 0.65 | |
| Ala-MCA | Wild type | + | 930 ± 150 | 1.54 ± 0.20 | 1.65 ± 0.070 |
| G357H | + | 1159 ± 120 | 3.01 ± 0.26 | 2.60 ± 0.042 |
a Values are the mean ± S.D. (n = 4).