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

摘要

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 a-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).