Plant Physiol, April 2000, Vol. 122, pp. 1417-1426

Purified -Glutamyl Transpeptidases from Tomato Exhibit High Affinity for Glutathione and Glutathione S-Conjugates¹

Climate Stress Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705

-Glutamyl transpeptidases (GTases) are the only enzymes known to hydrolyze the unique N-terminal amide bonds of reduced glutathione (-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two GTases (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, -linked Glu and the artificial substrates -L-glutamyl-p-nitroanilide and -L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. GTase 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 GTases 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.