Table 1.
Hydrolysis of Tos-Gly-Pro-Arg-AMC by mutant trypsinogens
| Enzyme | kcat/Km (M−1s−1) | ΔΔG (kcal/mol) | Ki (μM) | ΔΔGBPTI (kcal/mol) |
| Wild-type trypsinc | 9 × 106 | — | 0.00044b | — |
| S195A trypsinogenc | NA | NA | ≥300a | ≥8 |
| I16G trypsinogenc | 0.069 | 11.0 | 110a | 7.3 |
| ΔI16V17 trypsinogenc | 0.025 | 11.6 | 9a | 5.9 |
| K15A trypsinogenc | 4.4 | 8.6 | 12.4b | 6.0 |
| ΔI16V17/D194N trypsinogenc | 3.9 | 8.6 | 12a (29)b | 6.0 |
| ΔI16V17/Q156K trypsinogen | 0.22 | 10.3 | 65b | 7.0 |
Michaelis-Menten parameters are listed below for the hydrolysis of Tos-Gly-Pro-Arg-AMC by wild-type and mutant trypsinogens. Values reported are the average of at least two independent experiments ± SEM. Assays were performed in 50 mM Hepes, pH 8.0, 100 mM NaCl and 10 mM CaCl2 at 25°C. ΔΔG is calculated from the ratio of kcat/Km for mutant to wild-type trypsin. The values of Kd for the dissociation of BPTI complexes were determined either by aisothermal titration calorimetry or bmonitoring the inhibition of enzyme activity. cData from Pasternak et al (Pasternak et al. 1998). ΔΔGBPTI is determined from the ratio of the Ki's of the mutant trypsinogens to trypsin.