Glutathione production by glutathione‐degrading enzymes displayed on proteoliposomes reconstituted from bovine kidney brush border membranes

Glutathione ( L ‐γ‐glutamyl‐ L ‐cysteinylglycine) is physiologically synthesized through two ATP‐dependent reactions catalyzed by γ‐glutamylcysteine synthase and glutathione synthase. The present study was designed to produce glutathione without the aid of ATP by using glutathione‐degrading enzymes, γ‐glutamyl transpeptidase and aminopeptidase M, in reverse: intrinsically the former enzyme catalyzes the cleavage of glutathione to give L ‐cysteinylglycine and a γ‐glutamyl moiety and the latter hydrolyzes the peptide linkage of L ‐cysteinylglycine. Both enzymes were simultaneously displayed on proteoliposomes, which were reconstituted from bovine kidney brush border membranes by a cholate dialysis method. The kinetic analysis using artificial substrates, L ‐γ‐glutamyl‐ p ‐nitroanilide for γ‐glutamyl transpeptidase and L ‐leucine‐ p ‐nitroanilide for aminopeptidase M, revealed that the proteoliposome reconstitution significantly increased the enzyme activities: for both the enzymes the maximum reaction rates were increased and Michaelis constants with the respective substrates were decreased. When the proteoliposomes were incubated with the amino acids glycine, L ‐cysteine, and L ‐glutamate (or L ‐glutamine) at 37 °C, a new product was determined on HPLC analyses using ODS and cation‐exchange columns, coinciding in retention time with authentic glutathione. This product was identified to be glutathione by LC–MS and 1 H‐NMR, after being purified by gel filtration using Sephadex G10 and HSKgel Toyopearl HW‐40F in succession. When the incubation mixture contained acivicin and bestatin, specific inhibitors for γ‐glutamyl transpeptidase and aminopeptidase M, respectively, glutathione was not produced at all. These results indicated that glutathione was produced by two‐step reversible reactions of aminopeptidase M and γ‐glutamyl transpeptidase from its constituent amino acids. The equilibrium glutathione concentration obtained with L ‐glutamine as a glutamyl donor substrate was about 3.5 times higher than that obtained with L ‐glutamate. The maximum pH for the glutathione production was 7.0–7.5, reflecting pH dependence of the activities of the enzymes. Copyright © 2004 Society of Chemical Industry