TABLE 1.
Kinetic parameters of KLK2 toward synthetic substrates
For the Michaelis constants Km and the turnover number kcat, the weighted S.E. values of the non-linear regression fitting were used, whereas the error of the catalytic efficiency was calculated according to the formula given by Fenner (76).
| KLK2 sourcea | Substrate | Km | kcat | kcat/Km |
|---|---|---|---|---|
| μm | s−1 | m−1 s−1 | ||
| E. coli | Ac-OFR-AMCb | 45 ± 2 | 196.60 ± 5.04 | 4,368,890 ± 224,160 |
| LEXSY | Ac-OFR-AMC | 224 ± 11 | 96.05 ± 3.97 | 308,260 ± 27,520 |
| E. coli | H-PFR-AMCc | 69 ± 3 | 17.03 ± 0.14 | 246,810 ± 10,920 |
| LEXSY | H-PFR-AMC | 378 ± 27 | 13.57 ± 0.54 | 35,890 ± 2940 |
| E. coli | Bz-PFR-pNAc | 75 ± 2 | 5.77 ± 0.09 | 76,930 ± 2380 |
| LEXSY | Bz-PFR-pNA | 448 ± 44 | 2.59 ± 0.17 | 5780 ± 680 |
| E. coli | Mco-d-Nle-GR-pNAd | 302 ± 14 | 0.10 ± 0.01 | 330 ± 40 |
| LEXSY | Mco-d-Nle-GR-pNA | 3567 ± 317 | 1.95 ± 0.10 | 550 ± 60 |
a Expression system.
b Acetyl-Orn-Phe-Arg-AMC contains the non-proteinogenic amino acid ornithine in P3 position, which is a homolog of Lys, being shorter by one methylene group.
c The values for KLK2e with H-PFR-AMC and Bz-PFR-pNA correspond to the data of Skala et al. (9) but were corrected according to a new active site titration with PPACK, in which KLK2e was 15.5% active, whereas the LEXSY protein showed 45.3% activity.
d Mco-d-Nle-Gly-Arg-pNA, also known as Chromozym X, has the non-natural d-norleucine in the P3 position, whereas its side chain occupies the S4 rather than the S3 subsite.