Purified γ-Glutamyl Transpeptidases from Tomato Exhibit High Affinity for Glutathione and GlutathioneS-Conjugates

Abstract

Abstract γ-Glutamyl transpeptidases (γGTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (γ-l-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two γGTases (I and II) with K  m values for glutathione of 110 and 90 μm were purified 2,977-fold and 2,152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyze dipeptides and other tripeptides with N-terminal, γ-linked Glu and the artificial substrates γ-l-glutamyl-p-nitroanilide and γ-l-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, includingl-Met, l-Phe, l-Trp,l-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. γGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 m NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, Dye Matrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two γGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kD.