On the Mechanism of Action of Pseudolysin: Kinetic Study of the Enzymatic Condensation of Z-Ala with Phe-NH₂

Abstract

We previously demonstrated that pseudolysin, a neutral metalloproteinase from thermolysin family, is a suitable catalyst for peptide bond formation through reverse proteolysis. In order to get an insight into the yield- and rate-controlling factors, we studied the kinetics and mechanism of pseudolysin-catalysed synthesis. The condensation of Z-Ala-Phe-NH₂ from Z-Ala and Phe-NH₂, performed in a semi-organic homogeneous system, was chosen as a reaction model. We applied the conductimetric method for measuring the initial rates of the enzymatic reaction before product precipitation. We have both optimised synthesis and conductimetric conditions. Highest velocities were obtained in the pH range 7.0-7.5. When kept below 0.15, ionic strength did not significantly alter the rate values. Kinetic studies were then performed for varied Z-Ala and Phe-NH₂ concentrations at different, fixed concentrations of the alternative substrate, at pH 7.3 in 35% MeOH and for an ionic strength below 0.15. Graphical inspection and numerical analysis of initial-rate data permitted calculation of the kinetic parameters and proposition of a kinetic mechanism for pseudolysin. The values of kinetic constants were: 6.5 mM Z-Ala-Phe-NH₂ synthesised/min/μM pseudolysin (V_m), 70 mM (K_M of Z-Ala) and 180mM (K_M of Phe-NH₂). In addition, the condensation reaction proceeded via a rapid-equilibrium random bireactant mechanism, as was previously demonstrated for thermolysin, another extensively studied metalloprotease.

Key Words:

• Metalloproteases
• Pseudolysin
• Enzymatic synthesis
• Dipeptide derivatives
• Random bireactant mechanism