Two-step model (Fig 5) for mesotrypsin inhibition by APPI homodimers provides an excellent fit to experimental data.

Fitting the reaction velocity ( Eq 18 ) measured at different substrate concentration, allows us to extract the equilibrium inhibition constant K 2I . Each of the four plots shows the fit corresponding the measurements performed at a given APPI homodimer concentration [ I ], where [APPI Active Site] = 2[ I ], stands for the concentration of APPI monomer units. The added mesotrypsin concentration was maintained at [ E tot ] = 0.25 nM, and we adopted K I = 122.5 nM, as independently determined from the measurements performed with monomeric APPI ( Fig 4B ). The fitted values of K 2I are presented above each plot. Raw data can be found in SI.