Purification and Characterization of Tomato Leaf (Lycopersicon esculentum Mill.) Hydroperoxide Lyase

Hydroperoxide lyase (HPOOH lyase) was extracted from tomato leaves ( Lycopersicon esculentum Mill.) and purified to apparent homogeneity by fractionated precipitation with polyethylene glycol 6000, ion exchange chromatography, and ultrafiltration. The enzyme is a trimer of 73 000 Da units with a molecular mass of 216 000 (determined by native−PAGE and gel filtration); its p I is around 4.9. Enzyme activity measurments realized with 9- and 13-hydroperoxides of linoleic acid (9-La OOH and 13-La OOH, respectively), α-linolenic acid (9-Ln OOH and 13-Ln OOH, respectively), and γ-linolenic acid (9-γLn OOH and 13-γ-Ln OOH, respectively) revealed a great affinity for 13-Ln OOH. The enzyme is rapidly inhibited by its substrate (13-Ln OOH), but preincubation with the other five hydroperoxides, which are not degraded by the enzyme, also resulted in activity inhibition. Dialysis could not restore the activity. When 13-Ln OOH is reduced in its corresponding alcohol or converted to its methyl ester, the inhibition is reduced. Keywords: Tomato leaves; Lycopersicon esculentum Mill.; hydroperoxide lyase; linolenic acid hydroperoxide; linoleic acid hydroperoxide