Enzymatic synthesis of theanine with Escherichia coli γ-glutamyltranspeptidase from a series of γ-glutamyl anilide substrate analogues

In order to investigate the catalytic mechanism of Escherichia coli gamma-glutamyltranspeptidase, ten para- and meta-substituted gamma-glutamyl anilides were chemically prepared and employed as substrates to synthesize L-theanine to assay the activity of gamma-glutamyltranspeptidase. The reaction was optimized for gamma-glutamyl-p-nitroanilide. Key factors such as substrate specificity, pH, temperature, and the substrate mole ratio were all investigated. Kinetic studies of the acyl transfer reaction were described and the Hammett plot was constructed. This study indicated that the ratelimiting acylation reaction of gamma-glutamyltranspeptidase can apparently be accelerated by either the electron-withdrawing or electron-donating substituents of gamma-glutamyl anilides. The reaction could be catalyzed by the general acid and carboxy of Asp-433 or phenolic hydroxyl Tyr-444 may be the acid by autodock simulation for all prepared gamma-glutamyl anilides.