Mechanism of Dipeptidyl Carboxypeptidase Activity of Thermolysin

Article abstract of DOI:10.1246/bcsj.57.2965

Steady state and presteady state kinetic analyses were performed for thermolysin-catalysed hydrolysis of chromophoric tripeptide substrates with free carboxyl terminal.The pH dependence of k_cat showed fairly higher pK_a (ca.7) than that of the second-order rate parameter (k_cat/K_m)(ca.5) and the pH-dependence of K_m resembled that of K_i for N-blocked dipeptidyl inhibitor observed before (S.Kunigi 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.