Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin.
The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.
D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism*
D010455:Peptides; D013820:Thermolysin