Mechanism of Dipeptidyl Carboxypeptidase Activity of Thermolysin

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Abstract
Steady state and presteady state kinetic analyses were performed for thermolysin-catalyzed hydrolysis of chromophoric tripeptide substrates with free carboxyl terminal. The pH dependence of kcat showed fairly higher pKa (\simeq7) than that of the second-order rate parameter(kcat⁄Km)(\simeq5) and the pH-dependence of Km resembled that of Ki for N-blocked dipeptidyl inhibitor observed before (S. Kunugi et al., Eur. J. Biochem. 124, 157 (1982)). These findings indicated that the reaction with this type of substrate involves a nonproductive binding mode. In a presteady state kinetic study by stopped-flow method, a burst process of 10–20 ms order was observed before the linear steady state at a relatively low pH and temperature. This process showed moderate pH dependence. Considering these experimental results coupled with those accumulated so far on this enzyme, a unified mechanism including a nonproductive binding and an isomerization process in prior to the cleavage of the peptide bond was proposed for the dipeptidyl carboxypeptidase activity of this enzyme.