Table 1. Specificity constants of TGase-mediated transamidation.
0.2–0.8 μM mRuby2-Qtag test proteins and 5 mM Gly-OMe were reacted in the presence of 0.1 U TGase. Initial rates were measured over 20 min, using the GDH-coupled assay. Specificity constants were determined by fitting initial rate data to the Michaelis-Menten equation (see Materials and methods).
| Protein substrate | kcat/KM (μM-1 min-1) |
||
|---|---|---|---|
| bTG | mTG | TG2 | |
| mRuby2-YAHQAHY | 19 ± 3 | 34 ± 3 | 7 ± 1 |
| mRuby2-YAHQPHY | 13 ± 1 | 4.0 ± 0.2 | 4.0 ± 0.2 |
| mRuby2-YPHQPHY | 5 ± 2 | 3 ± 1 | 8 ± 5 |
| mRuby2-YPHQAHY | 8 ± 2 | 13 ± 3 | 1.0 ± 0.5 |
| mRuby2-YSHQAHY | 4 ± 1 | 0.20 ± 0.04 | 7 ± 2 |
| mRuby2-YAHQAAH | 2 ± 1 | 29 ± 2 | 2 ± 1 |
| mRuby2-7M48 | 7 ± 1 | 41 ± 4 | 9 ± 2 |
| MBP-RTQPA | 3.0 ± 0.4 | 7 ± 3 | 0.30 ± 0.04 |
| MBP-RLQQP | 1.0 ± 0.1 | 6 ± 2 | 1.0 ± 0.5 |