Table 3. Effect of N-terminal succinylation of a peptide substrate on the enzyme kinetic parameters of CTRB1, CTRB2, bCTRA, and CTRB1 mutants D236R and S242T.
The two Ala-Ala-Pro-Phe-p-nitroanilide (AAPF-pNA) substrates differ only at their N-terminus, where the succinylated (Suc-AAPF-pNA) version is negatively charged, while the free version (H-AAPF-pNA) is positively charged. The kcat and KM values are averages of two independent measurements, as described in the Materials and Methods.
| CTRB1 | CTRB2 | bCTRA | CTRB1 D236R | CTRB1 S242T | |
|---|---|---|---|---|---|
| Suc-AAPF-pNA | |||||
| kcat (s−1) | 34.6 | 26.1 | 36.9 | 36.0 | 38.4 |
| KM (μM) | 219 | 20.4 | 46.6 | 17.9 | 220 |
| kcat/KM (M−1s−1) | 1.6 x 105 | 1.3 x 106 | 7.9 x 105 | 2.0 x 106 | 1.8 x 105 |
|
| |||||
| H-AAPF-pNA | |||||
| kcat (s−1) | 22.8 | 10.9 | 20.3 | 9.64 | 21.3 |
| KM (μM) | 575 | 304 | 371 | 214 | 550 |
| kcat/KM (M−1s−1) | 4.0 x 104 | 3.6 x 104 | 5.5 x 104 | 4.5 x 104 | 3.9 x 104 |