-Carboxylic Acid-Conjugating Activity in Tomato Fruit

A new conjugate, 1 -(y-i-glutamylamino)cyclopropane-1 -carboxylic acid (CACC), of the ethylene precursor 1 -aminocyclopropane1 -carboxylic acid (ACC) i s identified. The only previously identified conjugate of ACC i s 1 -(malonylamino)cyclopropane-1 -carboxylic acid (MACC). CACC, not MACC, was the major conjugate formed by crude protein extracts of tomato (Lycopersicon esculentum Mil1 cv Ailsa Craig) fruit pericarp and seeds incubated with [14C]ACC. CACC was resolved from [14C]ACC and [14C]MACC by reversedphase C, thin-layer chromatography and subsequently detected and quantified using a radioisotope-imaging system. Proteins precipitated from crude extracts failed to catalyze formation of CACC unless the supernatant was added back. Reduced glutathione, but not other reducing agents, replaced the crude supernatant. When [35S-cysteine]glutathione and [3H-2-glycine]glutathione were used as substrates, neither radiolabeled glycine nor cysteine from the glutathione tripeptide was incorporated into CACC. Oxidized glutathione, S-substituted glutathione, and diand tripeptides having an N-terminal y-i-glutamic acid, but lacking cysteine and glycine, also served as substrates for CACC formation. Peptides lacking the N-terminal y-i-glutamic acid did not serve as substrates. Acid hydrolysis of CACC yielded ACC, suggesting that CACC is an amidelinked conjugate of ACC. Taken together, these results indicate that CACC is 1 -(y-glutamy1amino)cyclopropane-1 -carboxylic acid and that i t s formation i s catalyzed by a y-glutamyltranspeptidase. Cas chromatography-mass spectrometry analysis of the N-acetyl dimethyl ester of CACC confirmed this structure.