Transient kinetic studies of protein hydrolyses by endo- and exo-proteases on a 27 MHz quartz-crystal microbalance |
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Authors: | Furusawa Hiroyuki Takano Hiroki Okahata Yoshio |
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Institution: | Department of Biomolecular Engineering and Frontier Collaborative Research Centre, Tokyo Institute of Technology, B-53, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. |
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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 alpha-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). |
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