KI values of four ecotin orthologs, SGMI-2 and TFMI-3 towards all three MASP enzymes.

The ecotin-MASP affinities in Table 1 were measured at thermodynamic equilibrium as described in the Materials and methods. For tight-binding inhibition assays, enzyme and inhibitor concentrations need to be low, close to the KI value, and reaching the equilibrium requires several hours-long incubation. Then, small synthetic substrates are added, and their conversion rates report on the concentration of the free enzyme. In these short readout time assays the small substrates compete only with the relatively high-off-rate inhibitors and in this case the measured apparent KI value needs to be corrected with KM (see KM values in Fig 2). On the other hand, small substrates do not compete with the low off-rate tight-binding inhibitors for enzyme binding.