Fig. 4.

Peptidase assay for the HslV mutants considered. A fluorogenic peptide Z-GGL-AMC was used as a substrate. The V–substrate curves were fitted to a simple Michaelis–Menten equation (solid lines) as indicated. Note that mutations showing large chemical shift perturbations (Fig. 3 and SI Appendix, Fig. S6) also give rise to an order of magnitude change in activity (either increase or decrease), whereas mutants (for example, I159V) producing small chemical shift changes have little effect on activity. Values of k_cat/K_m (M⁻¹s⁻¹) are indicated. The values, including errors, are WT = 18 ± 2; L71V = 422 ± 8; L72V = 20 ± 4; V76A = 151 ± 10; L88I = 136 ± 5; L91I = 1.40 ± 0.03; V138I = 47 ± 5; I159V = 37 ± 8; and V161I = 58 ± 2.