Table IV.
Burst Kinetic Constants for Hydrolysis of Z-GPR-pNA in Presence of BPTI by Trypsin Variantsa
| Burst kinetic constants |
||||||
|---|---|---|---|---|---|---|
|
kobs (×10−2) |
||||||
| Variant | 1 µM BPTI | 2 µM BPTI | 4 µM BPTI | 10 µM BPTI | kon (µM−1 s−1) | koff (s−1) |
| WT | 0.87b | 1.25 ± 0.03 | 3.0 ± 0.1 | 1.3 ± 0.1 | 5.5 x 10−3 | 2.5 x 10−3 |
| Y39F | 0.88b | 1.61b | 3.57 ± 0.04 | 5.22 ± 0.2 | 6.7 x 10−3 | 2.4 x 10−3 |
| Y39A | 0.64 ± 0.04 | 1.18 ± 0.05 | 2.56 ± 0.03 | 4.4 ± 0.1 | 4.1 x 10−3 | 3.3 x 10−3 |
| K60V | 1.41 ± 0.01 | 3.5 ± 0.1 | 7.5 ± 0.4 | N/D | 1.48 x 10−2 | 2.0 x 10−4 |
| K60A | 1.23 ± 0.01 | 2.70 ± 0.05 | 5.5 ± 0.4 | N/D | 1.04 x 10−2 | 3.8 x 10−3 |
Standard deviation of these values was less than 1%.
a
% Free enzyme was determined at 25°C in 50 mM HEPES, 100 mM NaCl, 20 mM CaCl2 (pH 8.0) using the fraction of initial rates [Eq. (2)]. Errors were determined by four trials.
b
Errors of these values were less than 1%.
N/D = not detected; describes a signal that was less than three times that of the noise.