Table 2.
Kinetics parameters of mutant KLKs (KLK4 and KLK7) at pH 8.0.
| Enzyme | Active site configuration | kcat (s−1) | KM (µM) | kcat/KM (s−1M−1) | % Activity* | ||
|---|---|---|---|---|---|---|---|
| KLK7 | Ser195 | Asp102 | His57 | 24.5 ± 0.7 | 303.6 ± 20.16 | 8.08 × 104 | 100 |
| mKLK7 | Ala195 | Asp102 | His57 | 0.34 ± 0.09 | 429.6 ± 224.2 | 7.93 × 102 | 0.98 |
| dmKLK7 | Ala195 | Asn102 | His57 | N/A | N/A | N/A | N/A |
| KLK4 | Ser195 | Asp102 | His57 | 21.9 ± 0.6 | 40.53 ± 3.77 | 5.41 × 105 | 100 |
| mKLK4 | Ala195 | Asp102 | His57 | 0.618 ± 0.09 | 101 ± 41 | 6.13 × 103 | 1.13 |
| dmKLK4 | Ala195 | Asn102 | His57 | N/A | N/A | N/A | N/A |
Kinetic parameters for the active and mutant KLKs were measured using the MeO-Suc-Arg-Pro-Tyr-MCA (for KLK7) and d-VLR-AFC (for KLK4) substrates. KM and kcat values were calculated using the nonlinear regression analysis in Graphpad Prism (n = 6). Velocity values for dmKLK7 and dmKLK4 did not fit the Michaelis-Menten curve suggesting no residual activity and indicated as N/A. Data are presented as ±standard error. *Percentage activity of mKLKs is calculated relative to the catalytic activity of KLKs with MeO-Suc-Arg-Pro-Tyr-MCA or d-VLR-AFC substrates.