Transient kinetic studies of protein hydrolyses by endo- and exo-proteases on a 27 MHz quartz-crystal microbalance

Abstract

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