Fig. 7

Michaelis-Menten analysis of TtSlyD at 25 °C. The data for full-length TtSlyD (TtSlyD_FL are shown as black filled circles, and the data for TtSlyD constructs with the insert-in-flap (IF) domain replaced by the flap loop from human FKBP12 (TtSlyD_ΔIF) are shown as gray open circles. a Determination of k_cat/K_M using the suc-ALPF-pNA tetrapeptide as substrate. The enzyme concentrations were varied, while 500 μM suc-ALPF-pNA (<< K_M) was used in all experiments. We determined k_cat/K_M to be 1.47 ± 0.05 • 10⁶ M⁻¹s⁻¹ for TtSlyD_FL and 0.85 ± 0.01 • 10⁶ M⁻¹s⁻¹ for TtSlyD_ΔIF, using linear regression analysis. b Full Michaelis-Menten analysis using the S2-P25A peptide as substrate. A quantity of 0.2 μM of enzyme was used in all experiments, while the peptide concentration was varied. By using the previously determined cis content of 14.5 %, these concentrations were transformed into concentrations of S2-P25A cis. Michaelis-Menten fitting of TtSlyD_FL resulted in a k_cat/K_M of 3.68 ± 0.04 • 10⁸ M⁻¹s⁻¹ with k_cat = 740,000 ± 140,000 s⁻¹ and K_M = 2000 ± 410 μM. Because less curvature and precision was obtained for TtSlyD_ΔIF, only the k_cat/K_M of 2.84 ± 0.01 • 10⁸ M⁻¹s⁻¹ is reported