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

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 degrees C up to 110 degrees C. The stabilization was caused by the interac tion 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 consta nt following the temperature elevation of the medium. The binding of the en zyme to Phe-OMe suggested the induction of the transition state formation a t around 80 degrees C based on the UV spectra, resulting in the increase in the stability in the higher temperature region. The fluorescence second-or der derivative spectra suggested that the binding Phe-OMe interacted with T rp 115 at the active site of the enzyme. The phenomenon was considered to b e a novel stabilization pattern of the enzyme resulting from the conduction due to the chemical modification by the binding substrate.