γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.
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