Figure 4.

Tight-binding titration of furin by α₁-PDX/hf and Dec-RVKR-CH₂Cl. Furin/f (5.4 nM) was incubated with increasing amounts of α₁-PDX/hf (○) and Dec-RVKR-CH₂Cl (▪) for 30 min at room temperature. pERTKR-MCA (100 μM) was added to determine residual furin activity. Because valid titration curves can be obtained for tight-binding titrants when [E]₀/K_i > 2 (24), the K_i of α₁-PDX/hf for furin was obtained by fitting the data to an equation for equilibrium binding (24). This analysis revealed a value of K_i = 0.60 nM and a value of [E]₀ = 10 nM. Importantly, the calculated ratio of [E]₀/K_i = 17 confirmed the validity of the K_i value obtained by using α₁-PDX/hf as tight-binding titrants of furin. (Inset) The data shown were used to determine the stoichiometry of the reaction between α₁-PDX/hf and furin. The abscissa shows the inhibitor/enzyme ratio using a value for active furin ([E]₀ = 5.4 nM) determined by titration using the active-site-directed irreversible inhibitor Dec-RVKR-CH₂Cl. The data are averages of duplicate samples and are representative of three independent experiments.