We have compared endo- and exo-type protease reactions and characterized the enzymatic reaction mechanisms by determining all kinetic parameters (kon, koff, kcat, Kd = koff/kon, and Km = (koff + kcat)/kon) by following the mass change of the formation and the decay of the enzyme–substrate (ES) complex (kon and koff), and the formation of the product (kcat) on a 27 MHz quartz-crystal microbalance in aqueous solutions. The Km value was nearly equal to the Kd value for the endo-type protease (subtilisin and α-chymotrypsin); however, in the case of exo-type protease (carboxypeptidase P), the Km value was quite different from the Kd value, due to kcat ≫ koff.
