Kinetic analysis of SCCA1 hinge mutants. The effect of hinge mutations on the inhibition of catS was measured under pseudo-first-order conditions by using the progress-curve method. Wild-type SCCA1 (□), SCCA1 AlaP14Thr mutant (■), or SCCA1 AlaP14Arg mutant (•) were incubated with catS and substrate at 25°C in cathepsin buffer, pH 5.5. The progress of catS inactivation was followed by measuring the relative fluorescence (RFU) over time. The rate of inhibition of catS by the AlaP14Thr SCCA1 mutant was kass = 3.9 × 105 M−1⋅s−1. CatS without inhibitor (○) served as a control. A set of curves from a representative experiment is depicted. Concentrations of reagents: inhibitor, 250 nM; enzyme, 10 nM; substrate [(Z-FR)2-R110], 5 μM.