Novel stabilization pattern of thermolysin due to the binding substrate induced by electrostatic change in enzyme active site caused by the temperature elevation

Abstract

During the synthesis of the dipeptide, N-benzyloxycarbonyl-l-phenylalanyl-l-phenylalanine methyl ester, from N-benzyloxycarbonyl-l-phenylalanine and l-phenylalanine methyl ester by thermolysin, the enzyme was stabilized by 20 °C up to 110 °C. The stabilization was caused by the interaction of the enzyme with Phe-OMe, a counterpart of the substrate, which was bound at the enzyme active site due to the drop in pH and dielectric constant following the temperature elevation of the medium. The binding of the enzyme to Phe-OMe suggested the induction of the transition state formation at around 80 °C based on the UV spectra, resulting in the increase in the stability in the higher temperature region. The fluorescence second-order derivative spectra suggested that the binding Phe-OMe interacted with Trp 115 at the active site of the enzyme. The phenomenon was considered to be a novel stabilization pattern of the enzyme resulting from the conduction due to the chemical modification by the binding substrate.